MicroRNA fingerprints during human megakaryocytopoiesis

ABSTRACT

The present invention provides novel methods and compositions for the diagnosis, prognosis and treatment of cancer and myeloproliferative disorders. The invention also provides methods of identifying anti-cancer agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of parent U.S. patentapplication Ser. No. 12/293,472, having a 371 filing date of Oct. 9,2008, now allowed, which is a national stage application filed under 37CFR§1.371 of international application PCT/US2007/006824 filed Mar. 19,2007, which claims the priority to U.S. Provisional Application Ser. No.61/743,585 filed Mar. 20, 2006, the entire disclosures of which areexpressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was supported, in whole or in part, by National Institutesof Health Program Project Grants PO1CA76259, PO1CA16058, PO1CA81534 andP01CA16672. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are a small non-coding family of 19-25 nucleotideRNAs that regulate gene expression by targeting messenger RNAs (mRNA) ina sequence specific manner, inducing translational repression or mRNAdegradation depending on the degree of complementarity between miRNAsand their targets (Bartel, D. P. (2004) Cell 116, 281-297; Ambros, V.(2004) Nature 431, 350-355). Many miRNAs are conserved in sequencebetween distantly related organisms, suggesting that these moleculesparticipate in essential processes. Indeed, miRNAs are involved in theregulation of gene expression during development (Xu, P., et al. (2003)Curr. Biol. 13, 790-795), cell proliferation (Xu, P., et al. (2003)Curr. Biol. 13, 790-795), apoptosis (Cheng, A. M., et al. (2005) Nucl.Acids Res. 33, 1290-1297), glucose metabolism (Poy, M. N., et al. (2004)Nature 432, 226-230), stress resistance (Dresios, J., et al. (2005)Proc. Natl. Acad. Sci. USA 102, 1865-1870) and cancer (Calin, G. A, etal. (2002) Proc. Natl. Acad. Sci. USA 99, 1554-15529; Calin, G. A., etal. (2004) Proc. Natl. Acad. Sci. USA 101, 11755-11760; He, L., et al.(2005) Nature 435, 828-833; and Lu, J., et al. (2005) Nature435:834-838).

There is also strong evidence that miRNAs play a role in mammalianhematopoiesis. In mice, miR-181, miR-223 and miR-142 are differentiallyexpressed in hematopoietic tissues, and their expression is regulatedduring hematopoiesis and lineage commitment (Chen, C. Z., et al. (2004)Science 303, 83-86). The ectopic expression of miR-181 in murinehematopoietic progenitor cells led to proliferation in the B-cellcompartment (Chen, C. Z., et al. (2004) Science 303, 83-86). SystematicmiRNA gene profiling in cells of the murine hematopoietic systemrevealed different miRNA expression patterns in the hematopoietic systemcompared with neuronal tissues, and identified individual miRNAexpression changes that occur during cell differentiation (Monticelli,S., et al. (2005) Genome Biology 6, R71). A recent study has identifieddown-modulation of miR-221 and miR-222 in human erythropoietic culturesof CD34⁺ cord blood progenitor cells (Felli, N., et al. (2005) Proc.Natl. Acad. Sci. USA. 102, 18081-18086). These miRNAs were found totarget the oncogene c-Kit. Further functional studies indicated that thedecline of these two miRNAs in erythropoietic cultures unblocks Kitprotein production at the translational level leading to expansion ofearly erythroid cells (Felli, N., et al. (2005) Proc. Natl. Acad. Sci.USA. 102, 18081-18086). In line with the hypothesis of miRNAs regulatingcell differentiation, miR-223 was found to be a key member of aregulatory circuit involving C/EBPa and NFI-A, which controlsgranulocytic differentiation in all-trans retinoic acid-treated acutepromyelocytic leukemic cell lines (Fazi, F., et al. (2005) Cell 123,819-831).

miRNAs have also been found deregulated in hematopoietic malignancies.Indeed, the first report linking miRNAs and cancer involved the deletionand down regulation of the miR-15a and miR-16-1 cluster, located atchromosome 13q14.3, a commonly-deleted region in chronic lymphocyticleukemia (Calin, G. A, et al. (2002) Proc. Natl. Acad. Sci. USA 99,1554-15529). High expression of miR-155 and host gene BIC was alsoreported in B-cell lymphomas (Metzler M., et al. (2004) GenesChromosomes and Cancer 39; 167-169). More recently it was shown that themiR-17-92 cluster, which is located in a genomic region of amplificationin lymphomas, is overexpressed in human B-cell lymphomas and theenforced expression of this cluster acted in concert with c-MYCexpression to accelerate tumor development in a mouse B cell lymphomamodel (He, L., et al. (2005) Nature 435, 828-833). These observationsindicate that miRNAs are important regulators of hematopoiesis and canbe involved in malignant transformation.

Platelets play an essential role in hemostasis and thrombosis. They areproduced from in large numbers from their parent cells, bone marrowmegakaryocytes, and arise from fragmentation of the cytoplasm. Onlyrecently has the molecular basis of what may turn out to be a largefamily of related disorders affecting platelet production started to bedefined. If the level of circulating platelets drops below a certainnumber (thrombocytopenia), the patient runs the risk of catastrophichemorrhage. Patients with cancer who have received chemotherapy or bonemarrow transplants usually have thrombocytopenia, and the slow recoveryof platelet count in these patients has been a concern. The demand forplatelet units for transfusion has been steadily increasing primarilybecause of the need to maintain a certain platelet level in suchpatients with cancer or those undergoing major cardiac surgery.

Identification of microRNAs that are differentially-expressed in cancercells (e.g., leukemia cells) may help pinpoint specific miRNAs that areinvolved in cancer and other disorders (e.g., platelet disorders).Furthermore, the identification of putative targets of these miRNAs mayhelp to unravel their pathogenic role. In particular, discovering thepatterns and sequence of miRNA expression during hematopoieticdifferentiation may provide insights about the functional roles of thesetiny non-coding genes in normal and malignant hematopoiesis.

There is a need for novel methods and compositions for the diagnosis,prognosis and treatment of cancer, myeloproliferative disorders andplatelet disorders (e.g., inherited platelet disorders).

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification ofspecific miRNAs that are involved in megakaryocytic differentiationand/or have altered expression levels in cancerous cells (e.g., in acutemegakaryoblastic leukemia (AMKL cell lines)). In the present study, themiRNA gene expression in human megakaryocyte cultures from bone marrowCD34⁺ progenitors and acute megakaryoblastic leukemia cell lines wasinvestigated. The results of this analysis indicate that several miRNAsare downregulated during normal megakaryocytic differentiation. Theresults further demonstrate that these miRNAs target genes involved inmegakaryocytopoiesis, while others are over expressed in cancer cells.

Accordingly, the invention encompasses methods of diagnosing orprognosticating cancer and/or a myeloproliferative disorder in a subject(e.g., a human). According to the methods of the invention, the level ofat least one miR gene product in a test sample from the subject iscompared to the level of a corresponding miR gene product in a controlsample. An alteration (e.g., an increase, a decrease) in the level ofthe miR gene product in the test sample, relative to the level of acorresponding miR gene product in the control sample, is indicative ofthe subject either having, or being at risk for developing, cancerand/or a myeloproliferative disorder. In one embodiment, the level ofthe miR gene product in the test sample from the subject is greater thanthat of the control. In another embodiment, the at least one miR geneproduct is selected from the group consisting of miR-101, miR-126,miR-99a, miR-99-prec, miR-106, miR-339, miR-99b, miR-149, miR-33,miR-135 and miR-20. In still another embodiment, the at least one miRgene product is selected from the group consisting of miR-101, miR-126,miR-106, miR-20 and miR-135. In yet another embodiment, the at least onemiR gene product is selected from the group consisting of miR-106,miR-20 and miR-135. In particular embodiments, the cancer that isdiagnosed or prognosticated is a leukemia (e.g., acute myeloid leukemia(e.g., acute megakaryoblastic leukemia)) or multiple myeloma. In otherembodiments, the myeloproliferative disorder is selected from the groupconsisting of essential thrombocytemia (ET), polycythemia vera (PV),myelodisplasia, myelofibrosis (e.g., agnogenic myeloid metaplasia (AMM)(also referred to as idiopathic myelofibrosis)) and chronic myelogenousleukemia (CML).

In another embodiment, the invention is a method of treating a cancerand/or a myeloproliferative disorder in a subject (e.g., a human). Inthe method, an effective amount of a compound for inhibiting expressionof at least one miR gene product selected from the group consisting ofmiR-101, miR-126, miR-99a, miR-99-prec, miR-106, miR-339, miR-99b,miR-149, miR-33, miR-135 and miR-20 is administered to the subject. Inone embodiment, the compound for inhibiting expression of at least onemiR gene product inhibits expression of a miR gene product selected fromthe group consisting of miR-101, miR-126, miR-106, miR-20 and miR-135.In another embodiment, the compound for inhibiting expression of atleast one miR gene product inhibits expression of a miR gene productselected from the group consisting of miR-106, miR-20 and miR-135. Inparticular embodiments, the cancer that is treated is a leukemia (e.g.,acute myeloid leukemia (e.g., acute megakaryoblastic leukemia)) ormultiple myeloma. In other embodiments, the myeloproliferative disorderis selected from the group consisting of essential thrombocytemia (ET),polycythemia vera (PV), myelodisplasia, myelofibrosis (e.g., agnogenicmyeloid metaplasia (AMM)) and chronic myelogenous leukemia (CML).

In another embodiment, the invention is a method of treating a cancerand/or a myeloproliferative disorder associated with overexpression of aMAFB gene product in a subject (e.g., a human). In the method, aneffective amount of at least one miR gene product or a variant orbiologically-active fragment thereof, which binds to, and decreasesexpression of, the MAFB gene product, is administered to the subject. Inone embodiment, the at least one miR gene product, variant orbiologically-active fragment thereof comprises a nucleotide sequencethat is complementary to a nucleotide sequence in the MAFB gene product.In another embodiment, the at least one miR gene product is miR-130a ora variant or biologically-active fragment thereof. Cancers andmyeloproliferative disorders suitable for treatment using this methodinclude, for example, those described herein.

In another embodiment, the invention is a method of treating a cancerand/or a myeloproliferative disorder associated with overexpression of aHOXA1 gene product in a subject (e.g., a human). In the method, aneffective amount of at least one miR gene product or a variant orbiologically-active fragment thereof, which binds to, and decreasesexpression of, the HOXA1 gene product, is administered to the subject.In one embodiment, the at least one miR gene product, variant orbiologically-active fragment thereof comprises a nucleotide sequencethat is complementary to a nucleotide sequence in the HOXA1 geneproduct. In another embodiment, the at least one miR gene product ismiR-10a or a variant or biologically-active fragment thereof. Cancersand myeloproliferative disorders suitable for treatment using thismethod include, for example, those described herein.

In one embodiment, the invention is a method of determining and/orpredicting megakaryocytic differentiation. In this method, the level ofat least one miR gene product in a sample (e.g., a sample from a subject(e.g., a human)) comprising megakaryocyte progeny and/or megakaryocytesis determined. That level is compared to the level of the correspondingmiR gene product in a control. An alteration in the level of the atleast one miR gene product in the sample, relative to that of thecontrol, is indicative of megakaryocytic differentiation. In oneembodiment, the alteration is a decrease in the level of the at leastone miR gene product in the sample. In another embodiment, the at leastone miR gene product is selected from the group consisting of miR-10a,miR-126, miR-106, miR-010b, miR-130a, miR-130a-prec, miR-124a,miR-032-prec, miR-101, miR-30c, miR-213, miR-132-prec, miR-150, miR-020,miR-339, let-7a, let-7d, miR-181c, miR-181b and miR-017. In stillanother embodiment, the at least one miR gene product is selected fromthe group consisting of miR-10a, miR-10b, miR-30c, miR-106, miR-126,miR-130a, miR-132, and miR-143.

The invention further provides pharmaceutical compositions for treatingcancer and/or a myeloproliferative disorder. In one embodiment, thepharmaceutical compositions of the invention comprise at least one miRexpression-inhibition compound and a pharmaceutically-acceptablecarrier. In a particular embodiment, the at least one miRexpression-inhibition compound is specific for a miR gene product whoseexpression is greater in cancer cells (e.g., acute megakaryoblasticleukemia (AMKL) cells) than control cells (i.e., it is upregulated). Inone embodiment, the miR expression-inhibition compound is specific forone or more miR gene products selected from the group consisting ofmiR-101, miR-126, miR-99a, miR-99-prec, miR-106, miR-339, miR-99b,miR-149, miR-33, miR-135 and miR-20. In another embodiment, the miRexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting of miR-101, miR-126,miR-106, miR-20, and miR-135. In still another embodiment, the miRexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting of miR-106, miR-20 andmiR-135. In yet another embodiment, the pharmaceutical compositionfurther comprises at least one anti-cancer agent.

In one embodiment, the invention is a pharmaceutical composition fortreating a cancer associated with overexpression of a MAFB gene productand/or a myeloproliferative disorder associated with overexpression of aMAFB gene product. Such pharmaceutical compositions comprise aneffective amount of at least one miR gene product and apharmaceutically-acceptable carrier, wherein the at least one miR geneproduct binds to, and decreases expression of, the MAFB gene product. Inanother embodiment, the at least one miR gene product comprises anucleotide sequence that is complementary to a nucleotide sequence inthe MAFB gene product. In still another embodiment, the at least one miRgene product is miR-130a or a variant or biologically-active fragmentthereof. In yet another embodiment, the pharmaceutical compositionfurther comprises at least one anti-cancer agent.

In one embodiment, the invention is a pharmaceutical composition fortreating a cancer associated with overexpression of a HOXA1 gene productand/or a myeloproliferative disorder associated with overexpression of aHOXA1 gene product. Such pharmaceutical compositions comprise aneffective amount of at least one miR gene product and apharmaceutically-acceptable carrier, wherein the at least one miR geneproduct binds to, and decreases expression of, the HOXA1 gene product.In another embodiment, the at least one miR gene product comprises anucleotide sequence that is complementary to a nucleotide sequence inthe HOXA1 gene product. In still another embodiment, the at least onemiR gene product is miR-10a or a variant or biologically-active fragmentthereof. In yet another embodiment, the pharmaceutical compositionfurther comprises at least one anti-cancer agent.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D depict Northern Blots and Real Time miRNA-PCR results, whichvalidate microRNA chip data in CD34 progenitor differentiationexperiments.

FIG. 1A depicts Northern Blots for miR-130a, miR-10a and miR-223. Aloading RNA control was performed with U6.

FIG. 1B is a graph depicting RT-miRNA-PCR for miR-10a, miR-106, miR-126and miR-130a. miRNA expression is presented as fold difference withrespect to CD34⁺ cells before culture.

FIG. 1C is a graph depicting temporal expression of miR-223 bymicroarray.

FIG. 1D is a graph depicting temporal expression of miR-15-1 andmiR-16-1 by RT-miRNA PCR.

FIGS. 2A-2C demonstrate that MAFB is a target of miR-130a.

FIG. 2A depicts MAFB mRNA and protein expression data in CD34⁺progenitors induced to megakaryocytic differentiation. □-Actin was usedfor RT-PCR and Western blot loading controls.

FIG. 2B is a graph depicting relative repression of luciferase activityin MEG01 cells co-transfected with miR-10a and PGL3 3′UTR MAFB, miR-10awith PGL3 3′UTR, miR-10a seed match mutated and scramble with mutated,and wild type 3′UTR MAFB.

FIG. 2C depicts Western blots of MAFB total protein lysates in K562cells transfected with miR-130a and scramble.

FIGS. 3A-3G demonstrate that MiR-10a downregulates HOXA1 by mediatingRNA cleavage.

FIG. 3A is a graph depicting RT-PCR results for HOXA1 gene expression indifferentiated megakaryocytes (Relative amount of transcript withrespect to CD34⁺ progenitors at baseline).

FIG. 3B is a Western blot showing hoxa1 protein expression indifferentiated megakaryocytes.

FIG. 3C is a graph depicting relative repression of luciferase activityof HOXA1 3′ UTR cloned PGL3 reporter plasmid when co-transfected withmiR-10a and control scramble.

FIG. 3D is a schematic showing complementarity between miR-10a and theHOXA1 3′UTR as predicted by PICTAR.

FIG. 3E depicts RT-PCR results for miR-10a gene expression in scrambleand miR-10a precursor transfected K562 cells.

FIG. 3F depicts RT-PCR results for HOXA1 gene expression in scramble andmiR-10a precursor transfected K562 cells.

FIG. 3G is a Western blot showing HOXA1 expression in K562 cellstransfected with control scramble and precursor miR-10a.

FIGS. 4A and 4B show phenotypic characterization results of invitro-differentiated CD34⁺ progenitors.

FIG. 4A depicts May-Giemsa stains that were performed on cytospinpreparations from CD34⁺ progenitors in culture at different days ofculture (day 6, day 10, day 12 and day 14). At day 4, most of the cellswere immature, as evidenced by the high nucleous:cytoplasmic ratio.Larger and multinuclear cells were observed by day 10. At day 14,predominantly larger, polyploid cells with long cytoplasmic processesand numerous membrane blebs with invaginations and vacuoles (originalmagnification 400×) were observed.

FIG. 4B depicts FACS analysis of CD34 in vitro-differentiatedmegakaryocytes. The membrane phenotype of CD34⁺ progenitor cells thatare grown in culture is shown. Cells were harvested at days 10 (D+10),14 (D+14) and 16 (D+16) and were analyzed by single fluorescent labelingusing an anti-CD41 antibody, an anti-CD61a antibody, an anti-CD42aantibody and their respective isotype monoclonal antibodies (D+10isotype; D+14 isotype; D+16 isotype). Double labeling was performed withanti-CD41a and CD61b monoclonal Abs at day 14 only.

FIG. 5 is a graph depicting RT-PCR expression results for miR-20 andmiR-17 in differentiated megakaryocytes. The results are presented asfold difference with respect to CD34⁺ cells at baseline afternormalization with 18S and delta Ct calculations.

FIG. 6A is a graph depicting temporal expression of miR-16-1 duringmegakaryocytic differentiation. The absolute expression value ofmiR-16-1 was determined by a per-chip median normalization.

FIG. 6B is a graph depicting temporal expression of miR-142 duringmegakaryocytic differentiation. The absolute expression value of miR-142was determined by a per-chip median normalization.

FIG. 6C is a graph depicting temporal expression of miR-181b duringmegakaryocytic differentiation. The absolute expression value ofmiR-181b was determined by a per-chip median normalization.

FIG. 7 is a Northern Blot of total RNA obtained from K562 cellstransfected with miR-130a precursor and scramble sequences hybridizedwith the probe for miR-130a. An RNA loading control was performed usingU6 hybridization.

FIG. 8 is a schematic depicting microRNAs that are located in the HOXA,HOXB, HOXC and HOXD gene clusters.

FIG. 9A is a graph depicting HOXB4 gene expression in differentiatedmegakaryocytes. RT-PCR results for HOXB4 are shown as fold difference inthe expression level with respect to CD34⁺ progenitors at baseline(before culture).

FIG. 9B is a graph depicting HOXB5 gene expression in differentiatedmegakaryocytes. RT-PCR results for HOXB5 are shown as fold difference inthe expression levels with respect to CD34⁺ progenitors at baseline(before culture).

FIG. 10 is a graph depicting microRNA expression in acutemegakaryoblastic cell lines by RT-PCR. Results are expressed as folddifference with respect to CD34-differentiated megakaryocytes afternormalization with 18S and delta Ct calculations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based, in part, on the identification ofspecific microRNAs (miRNAs) that are involved in megakaryocyticdifferentiation and/or have altered expression levels in cancerous cells(e.g., in acute megakaryoblastic leukemia (AMKL cell lines)). Theinvention is further based, in part, on association of these miRNAs withparticular diagnostic, prognostic and therapeutic features. As describedand exemplified herein:

i) particular miRNA are downregulated during megakaryocyticdifferentiation;

ii) the transcription factor MAFB is a target for miR-130a;

iii) miR-10a expression parallels that of HOXB gene expression;

iv) miR-10a downregulates HOXA1 expression; and

v) particular miRNA are upregulated in cancerous cells (e.g., acutemegakaryoblastic leukemia (AMKL) cells).

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”“miR” or “miRNA” refers to the unprocessed or processed RNA transcriptfrom a miR gene. As the miR gene products are not translated intoprotein, the term “miR gene products” does not include proteins. Theunprocessed miR gene transcript is also called a “miR precursor,” andtypically comprises an RNA transcript of about 70-100 nucleotides inlength. The miR precursor can be processed by digestion with an RNAse(for example, Dicer, Argonaut, RNAse III (e.g., E. coli RNAse III)) intoan active 19-25 nucleotide RNA molecule. This active 19-25 nucleotideRNA molecule is also called the “processed” miR gene transcript or“mature” miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAse III). Itis understood that the active 19-25 nucleotide RNA molecule can also beproduced directly by biological or chemical synthesis, without having tobe processed from the miR precursor. When a microRNA is referred toherein by name, the name corresponds to both the precursor and matureforms, unless otherwise indicated.

Tables 1a and 1b depict the nucleotide sequences of particular precursorand mature human microRNAs.

TABLE 1a Human microRNA Precursor Sequences. Precursor SEQ ID NameSequence (5′ To 3′)* NO. let-7a-1 CACUGUGGGAUGAGGUAGUAGGUUGUAUAGUUUUAGGG1 UCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGU CUUUCCUAACGUG let-7a-2AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUUACAUC 2AAGGGAGAUAACUGUACAGCCUCCUAGCUUUCCU let-7a-3GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCC 3UGCUAUGGGAUAACUAUACAAUCUACUGUCUUUCCU let-7a-4GUGACUGCAUGCUCCCAGGUUGAGGUAGUAGGUUGUAU 4AGUUUAGAAUUACACAAGGGAGAUAACUGUACAGCCUC CUAGCUUUCCUUGGGUCUUGCACUAAACAAClet-7b GGCGGGGUGAGGUAGUAGGUUGUGUGGUUUCAGGGCAG 5UGAUGUUGCCCCUCGGAAGAUAACUAUACAACCUACUGC CUUCCCUG let-7cGCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUUAGAGU 6UACACCCUGGGAGUUAACUGUACAACCUUCUAGCUUUCC UUGGAGC let-7dCCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAGGGCAG 7GGAUUUUGCCCACAAGGAGGUAACUAUACGACCUGCUGC CUUUCUUAGG let-7d-v1CUAGGAAGAGGUAGUAGUUUGCAUAGUUUUAGGGCAAA 8GAUUUUGCCCACAAGUAGUUAGCUAUACGACCUGCAGCC UUUUGUAG let-7d-v2CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGGGUUGU 9GACAUUGCCCGCUGUGGAGAUAACUGCGCAAGCUACUGC CUUGCUAG let-7eCCCGGGCUGAGGUAGGAGGUUGUAUAGUUGAGGAGGAC 10ACCCAAGGAGAUCACUAUACGGCCUCCUAGCUUUCCCCA GG let-7f-1UCAGAGUGAGGUAGUAGAUUGUAUAGUUGUGGGGUAGU 11GAUUUUACCCUGUUCAGGAGAUAACUAUACAAUCUAUU GCCUUCCCUGA let-7f-2-1CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUGUGGGGUA 12GUGAUUUUACCCUGUUCAGGAGAUAACUAUACAAUCUA UUGCCUUCCCUGA let-7f-2-2CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUUUAGGGUC 13AUACCCCAUCUUGGAGAUAACUAUACAGUCUACUGUCUU UCCCACGG let-7gUUGCCUGAUUCCAGGCUGAGGUAGUAGUUUGUACAGUU 14UGAGGGUCUAUGAUACCACCCGGUACAGGAGAUAACUG UACAGGCCACUGCCUUGCCAGGAACAGCGCGClet-7i CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGGGUUGU 15GACAUUGCCCGCUGUGGAGAUAACUGCGCAAGCUACUGC CUUGCUAG miR-1b-1-1ACCUACUCAGAGUACAUACUUCUUUAUGUACCCAUAUGA 16ACAUACAAUGCUAUGGAAUGUAAAGAAGUAUGUAUUUU UGGUAGGC miR-1b-1-2CAGCUAACAACUUAGUAAUACCUACUCAGAGUACAUACU 17UCUUUAUGUACCCAUAUGAACAUACAAUGCUAUGGAAU GUAAAGAAGUAUGUAUUUUUGGUAGGCAAUAmiR-1b-2 GCCUGCUUGGGAAACAUACUUCUUUAUAUGCCCAUAUG 18GACCUGCUAAGCUAUGGAAUGUAAAGAAGUAUGUAUCU CAGGCCGGG miR-1bUGGGAAACAUACUUCUUUAUAUGCCCAUAUGGACCUGC 19UAAGCUAUGGAAUGUAAAGAAGUAUGUAUCUCA miR-1dACCUACUCAGAGUACAUACUUCUUUAUGUACCCAUAUGA 20ACAUACAAUGCUAUGGAAUGUAAAGAAGUAUGUAUUUU UGGUAGGC miR-7-1aUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUU 21UUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACA GUCUGCCAUAUGGCACAGGCCAUGCCUCUACAmiR-7-1b UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAU 22UUUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCAUGCCUCUACAG miR-7-2CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUGGAAGAC 23UAGUGAUUUUGUUGUUGUCUUACUGCGCUCAACAACAA AUCCCAGUCUACCUAAUGGUGCCAGCCAUCGCAmiR-7-3 AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGGAAGAC 24UAGUGAUUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCUCAUAGCGCAGACUCCCUUCGAC miR-9-1CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUGUAUGA 25GUGGUGUGGAGUCUUCAUAAAGCUAGAUAACCGAAAGU AAAAAUAACCCCA miR-9-2GGAAGCGAGUUGUUAUCUUUGGUUAUCUAGCUGUAUGA 26GUGUAUUGGUCUUCAUAAAGCUAGAUAACCGAAAGUAA AAACUCCUUCA miR-9-3GGAGGCCCGUUUCUCUCUUUGGUUAUCUAGCUGUAUGA 27GUGCCACAGAGCCGUCAUAAAGCUAGAUAACCGAAAGU AGAAAUGAUUCUCA miR-10aGAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAUCCGAA 28UUUGUGUAAGGAAUUUUGUGGUCACAAAUUCGUAUCUAGGGGAAUAUGUAGUUGACAUAAACACUCCGCUCU miR-10bCCAGAGGUUGUAACGUUGUCUAUAUAUACCCUGUAGAA 29CCGAAUUUGUGUGGUAUCCGUAUAGUCACAGAUUCGAUUCUAGGGGAAUAUAUGGUCGAUGCAAAAACUUCA miR-15a-2GCGCGAAUGUGUGUUUAAAAAAAAUAAAACCUUGGAGU 30AAAGUAGCAGCACAUAAUGGUUUGUGGAUUUUGAAAAG GUGCAGGCCAUAUUGUGCUGCCUCAAAAAUACmiR-15a CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGAU 31UUUGAAAAGGUGCAGGCCAUAUUGUGCUGCCUCAAAAA UACAAGG miR-15b-1CUGUAGCAGCACAUCAUGGUUUACAUGCUACAGUCAAG 32 AUGCGAAUCAUUAUUUGCUGCUCUAGmiR-15b-2 UUGAGGCCUUAAAGUACUGUAGCAGCACAUCAUGGUUU 33ACAUGCUACAGUCAAGAUGCGAAUCAUUAUUUGCUGCU CUAGAAAUUUAAGGAAAUUCAU miR-16-1GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUA 34AGAUUCUAAAAUUAUCUCCAGUAUUAACUGUGCUGCUG AAGUAAGGUUGAC miR-16-2GUUCCACUCUAGCAGCACGUAAAUAUUGGCGUAGUGAA 35AUAUAUAUUAAACACCAAUAUUACUGUGCUGCUUUAGU GUGAC miR-16-13GCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAU 36UCUAAAAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGU AAGGU miR-17GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUG 37AUAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUU AUGGUGAC miR-18UGUUCUAAGGUGCAUCUAGUGCAGAUAGUGAAGUAGAU 38UAGCAUCUACUGCCCUAAGUGCUCCUUCUGGCA miR-18-13UUUUUGUUCUAAGGUGCAUCUAGUGCAGAUAGUGAAGU 39AGAUUAGCAUCUACUGCCCUAAGUGCUCCUUCUGGCAUA AGAA miR-19aGCAGUCCUCUGUUAGUUUUGCAUAGUUGCACUACAAGA 40AGAAUGUAGUUGUGCAAAUCUAUGCAAAACUGAUGGUG GCCUGC miR-19a-13CAGUCCUCUGUUAGUUUUGCAUAGUUGCACUACAAGAA 41GAAUGUAGUUGUGCAAAUCUAUGCAAAACUGAUGGUGG CCUG miR-19b-1CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGC 42UGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGA CUGUGGUAGUG miR-19b-2ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUGCAUUU 43CAGCGUAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGC AAAACUGAUUGUGAUAAUGU miR-19b-13UUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGU 44GAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUG GUAG miR-19b-XUUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCGUAUAU 45AUGUAUAUGUGGCUGUGCAAAUCCAUGCAAAACUGAUU GUGAU miR-20GUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGUUUAGU 46 (miR-20a)UAUCUACUGCAUUAUGAGCACUUAAAGUACUGC miR-21UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAU 47CUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA miR-21-17ACCUUGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUU 48GAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACAU UUUG miR-22GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCUUUAUG 49UCCUGACCCAGCUAAAGCUGCCAGUUGAAGAACUGUUGC CCUCUGCC miR-23aGGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUG 50UCACAAAUCACAUUGCCAGGGAUUUCCAACCGACC miR-23bCUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAUGCUGAU 51UUGUGACUUAAGAUUAAAAUCACAUUGCCAGGGAUUAC CACGCAACCACGACCUUGGC miR-23-19CCACGGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUC 52CUGUCACAAAUCACAUUGCCAGGGAUUUCCAACCGACCC UGA miR-24-1CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUAC 53 ACACUGGCUCAGUUCAGCAGGAACAGGAGmiR-24-2 CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAGUUGGU 54UUGUGUACACUGGCUCAGUUCAGCAGGAACAGGG miR-24-19CCCUGGGCUCUGCCUCCCGUGCCUACUGAGCUGAAACAC 55AGUUGGUUUGUGUACACUGGCUCAGUUCAGCAGGAACA GGGG miR-24-9CCCUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUU 56ACACACUGGCUCAGUUCAGCAGGAACAGCAUC miR-25GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUUGCUGG 57ACGCUGCCCUGGGCAUUGCACUUGUCUCGGUCUGACAGU GCCGGCC miR-26aAGGCCGUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGU 58GCAGGUCCCAAUGGCCUAUCUUGGUUACUUGCACGGGGA CGCGGGCCU miR-26a-1GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGUGCAGG 59UCCCAAUGGGCCUAUUCUUGGUUACUUGCACGGGGACGC miR-26a-2GGCUGUGGCUGGAUUCAAGUAAUCCAGGAUAGGCUGUU 60UCCAUCUGUGAGGCCUAUUCUUGAUUACUUGUUUCUGG AGGCAGCU miR-26bCCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUG 61CUGUCCAGCCUGUUCUCCAUUACUUGGCUCGGGGACCGG miR-27aCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGGGUCCA 62CACCAAGUCGUGUUCACAGUGGCUAAGUUCCGCCCCCCA G miR-27b-1AGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGAUUGGU 63UUCCGCUUUGUUCACAGUGGCUAAGUUCUGCACCU miR-27b-2ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUUGGUGA 64ACAGUGAUUGGUUUCCGCUUUGUUCACAGUGGCUAAGU UCUGCACCUGAAGAGAAGGUG miR-27-19CCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGGGUCC 65ACACCAAGUCGUGUUCACAGUGGCUAAGUUCCGCCCCCC AGG miR-28GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUGAGUUAC 66CUUUCUGACUUUCCCACUAGAUUGUGAGCUCCUGGAGGG CAGGCACU miR-29a-2CCUUCUGUGACCCCUUAGAGGAUGACUGAUUUCUUUUG 67GUGUUCAGAGUCAAUAUAAUUUUCUAGCACCAUCUGAA AUCGGUUAUAAUGAUUGGGGAAGAGCACCAUGmiR-29a AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUU 68UUCUAGCACCAUCUGAAAUCGGUUAU miR-29b-1CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGAUUUAA 69AUAGUGAUUGUCUAGCACCAUUUGAAAUCAGUGUUCUU GGGGG miR-29b-2CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGAUUUUU 70CCAUCUUUGUAUCUAGCACCAUUUGAAAUCAGUGUUUU AGGAG miR-29cACCACUGGCCCAUCUCUUACACAGGCUGACCGAUUUCUC 71CUGGUGUUCAGAGUCUGUUUUUGUCUAGCACCAUUUGA AAUCGGUUAUGAUGUAGGGGGAAAAGCAGCAGCmiR-30a GCGACUGUAAACAUCCUCGACUGGAAGCUGUGAAGCCAC 72AGAUGGGCUUUCAGUCGGAUGUUUGCAGCUGC miR-30b-1AUGUAAACAUCCUACACUCAGCUGUAAUACAUGGAUUG 73 GCUGGGAGGUGGAUGUUUACGUmiR-30b-2 ACCAAGUUUCAGUUCAUGUAAACAUCCUACACUCAGCUG 74UAAUACAUGGAUUGGCUGGGAGGUGGAUGUUUACUUCA GCUGACUUGGA miR-30cAGAUACUGUAAACAUCCUACACUCUCAGCUGUGGAAAG 75UAAGAAAGCUGGGAGAAGGCUGUUUACUCUUUCU miR-30dGUUGUUGUAAACAUCCCCGACUGGAAGCUGUAAGACAC 76AGCUAAGCUUUCAGUCAGAUGUUUGCUGCUAC miR-30eCUGUAAACAUCCUUGACUGGAAGCUGUAAGGUGUUCAG 77 AGGAGCUUUCAGUCGGAUGUUUACAGmiR-31 GGAGAGGAGGCAAGAUGCUGGCAUAGCUGUUGAACUGG 78GAACCUGCUAUGCCAACAUAUUGCCAUCUUUCC miR-32GGAGAUAUUGCACAUUACUAAGUUGCAUGUUGUCACGG 79CCUCAAUGCAAUUUAGUGUGUGUGAUAUUUUC miR-33bGGGGGCCGAGAGAGGCGGGCGGCCCCGCGGUGCAUUGCU 80GUUGCAUUGCACGUGUGUGAGGCGGGUGCAGUGCCUCG GCAGUGCAGCCCGGAGCCGGCCCCUGGCACCACmiR-33b-2 ACCAAGUUUCAGUUCAUGUAAACAUCCUACACUCAGCUG 81UAAUACAUGGAUUGGCUGGGAGGUGGAUGUUUACUUCA GCUGACUUGGA miR-33CUGUGGUGCAUUGUAGUUGCAUUGCAUGUUCUGGUGGU 82ACCCAUGCAAUGUUUCCACAGUGCAUCACAG miR-34-aGGCCAGCUGUGAGUGUUUCUUUGGCAGUGUCUUAGCUG 83GUUGUUGUGAGCAAUAGUAAGGAAGCAAUCAGCAAGUAUACUGCCCUAGAAGUGCUGCACGUUGUGGGGCCC miR-34-bGUGCUCGGUUUGUAGGCAGUGUCAUUAGCUGAUUGUAC 84UGUGGUGGUUACAAUCACUAACUCCACUGCCAUCAAAAC AAGGCAC miR-34-cAGUCUAGUUACUAGGCAGUGUAGUUAGCUGAUUGCUAA 85UAGUACCAAUCACUAACCACACGGCCAGGUAAAAAGAUU miR-91-13UCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGA 86UAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUUA UGGUGA miR-92-1CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCUGUGUU 87UCUGUAUGGUAUUGCACUUGUCCCGGCCUGUUGAGUUU GG miR-92-2UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUGUGUUC 88UAUAUAAAGUAUUGCACUUGUCCCGGCCUGUGGAAGA miR-93-1CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAGUGUGA 89 (miR-93-2)UUACCCAACCUACUGCUGAGCUAGCACUUCCCGAGCCCC CGG miR-95-4AACACAGUGGGCACUCAAUAAAUGUCUGUUGAAUUGAA 90AUGCGUUACAUUCAACGGGUAUUUAUUGAGCACCCACUC UGUG miR-96-7UGGCCGAUUUUGGCACUAGCACAUUUUUGCUUGUGUCU 91CUCCGCUCUGAGCAAUCAUGUGCAGUGCCAAUAUGGGAA A miR-97-6GUGAGCGACUGUAAACAUCCUCGACUGGAAGCUGUGAA 92 (miR-30*)GCCACAGAUGGGCUUUCAGUCGGAUGUUUGCAGCUGCCU ACU miR-98GUGAGGUAGUAAGUUGUAUUGUUGUGGGGUAGGGAUAU 93UAGGCCCCAAUUAGAAGAUAACUAUACAACUUACUACU UUCC miR-99bGGCACCCACCCGUAGAACCGACCUUGCGGGGCCUUCGCC 94GCACACAAGCUCGUGUCUGUGGGUCCGUGUC miR-99aCCCAUUGGCAUAAACCCGUAGAUCCGAUCUUGUGGUGAA 95GUGGACCGCACAAGCUCGCUUCUAUGGGUCUGUGUCAGU GUG miR-100-1/2AAGAGAGAAGAUAUUGAGGCCUGUUGCCACAAACCCGU 96AGAUCCGAACUUGUGGUAUUAGUCCGCACAAGCUUGUA UCUAUAGGUAUGUGUCUGUUAGGCAAUCUCACmiR-100-11 CCUGUUGCCACAAACCCGUAGAUCCGAACUUGUGGUAUU 97AGUCCGCACAAGCUUGUAUCUAUAGGUAUGUGUCUGUU AGG miR-101-1/2AGGCUGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUGUC 98UAUUCUAAAGGUACAGUACUGUGAUAACUGAAGGAUGG CAGCCAUCUUACCUUCCAUCAGAGGAGCCUCACmiR-101 UCAGUUAUCACAGUGCUGAUGCUGUCCAUUCUAAAGGU 99 ACAGUACUGUGAUAACUGAmiR-101-1 UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUGUCUAUU 100CUAAAGGUACAGUACUGUGAUAACUGAAGGAUGGCA miR-101-2ACUGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUGUAU 101AUCUGAAAGGUACAGUACUGUGAUAACUGAAGAAUGGU GGU miR-101-9UGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUGUAUAU 102CUGAAAGGUACAGUACUGUGAUAACUGAAGAAUGGUG miR-102-1CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGAUUUUU 103CCAUCUUUGUAUCUAGCACCAUUUGAAAUCAGUGUUUU AGGAG miR-102-7.1CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGAUUUAA 104 (miR-102-7.2)AUAGUGAUUGUCUAGCACCAUUUGAAAUCAGUGUUCUU GGGGG miR-103-2UUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUGUAGCA 105UUCAGGUCAAGCAACAUUGUACAGGGCUAUGAAAGAAC CA miR-103-1UACUGCCCUCGGCUUCUUUACAGUGCUGCCUUGUUGCAU 106AUGGAUCAAGCAGCAUUGUACAGGGCUAUGAAGGCAUU G miR-104-17AAAUGUCAGACAGCCCAUCGACUGGUGUUGCCAUGAGA 107UUCAACAGUCAACAUCAGUCUGAUAAGCUACCCGACAAG G miR-105-1UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUGGUGGC 108UGCUCAUGCACCACGGAUGUUUGAGCAUGUGCUACGGU GUCUA miR-105-2UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUGGUGGC 109UGCUUAUGCACCACGGAUGUUUGAGCAUGUGCUAUGGU GUCUA miR-106-aCCUUGGCCAUGUAAAAGUGCUUACAGUGCAGGUAGCUU 110UUUGAGAUCUACUGCAAUGUAAGCACUUCUUACAUUAC CAUGG miR-106-bCCUGCCGGGGCUAAAGUGCUGACAGUGCAGAUAGUGGU 111CCUCUCCGUGCUACCGCACUGUGGGUACUUGCUGCUCCA GCAGG miR-107CUCUCUGCUUUCAGCUUCUUUACAGUGUUGCCUUGUGGC 112AUGGAGUUCAAGCAGCAUUGUACAGGGCUAUCAAAGCA CAGA MIR-108-1-ACACUGCAAGAACAAUAAGGAUUUUUAGGGGCAUUAUG 113 SMALLACUGAGUCAGAAAACACAGCUGCCCCUGAAAGUCCCUCA UUUUUCUUGCUGU MIR-108-2-ACUGCAAGAGCAAUAAGGAUUUUUAGGGGCAUUAUGAU 114 SMALLAGUGGAAUGGAAACACAUCUGCCCCCAAAAGUCCCUCAU UUU miR-122a-1CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUG 115UCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUAC UGCUAGGC miR-122a-2AGCUGUGGAGUGUGACAAUGGUGUUUGUGUCCAAACUA 116 UCAAACGCCAUUAUCACACUAAAUAGCUmiR-123 ACAUUAUUACUUUUGGUACGCGCUGUGACACUUCAAAC 117UCGUACCGUGAGUAAUAAUGCGC miR-124a-1AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUGAUUUAA 118AUGUCCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAA UGGGGCUG miR-124a-2AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCACAGCGGA 119CCUUGAUUUAAUGUCAUACAAUUAAGGCACGCGGUGAA UGCCAAGAGCGGAGCCUACGGCUGCACUUGAAGmiR-124a-3 UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUUGAUUUA 120AUGUCUAUACAAUUAAGGCACGCGGUGAAUGCCAAGAG AGGCGCCUCC miR-124aCUCUGCGUGUUCACAGCGGACCUUGAUUUAAUGUCUAU 121ACAAUUAAGGCACGCGGUGAAUGCCAAGAG miR-124bCUCUCCGUGUUCACAGCGGACCUUGAUUUAAUGUCAUAC 122 AAUUAAGGCACGCGGUGAAUGCCAAGAGmiR-125a-1 UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACCUGUGAG 123GACAUCCAGGGUCACAGGUGAGGUUCUUGGGAGCCUGG CGUCUGGCC miR-125a-2GGUCCCUGAGACCCUUUAACCUGUGAGGACAUCCAGGGU 124 CACAGGUGAGGUUCUUGGGAGCCUGGmiR-125b-1 UGCGCUCCUCUCAGUCCCUGAGACCCUAACUUGUGAUGU 125UUACCGUUUAAAUCCACGGGUUAGGCUCUUGGGAGCUG CGAGUCGUGCU miR-125b-2ACCAGACUUUUCCUAGUCCCUGAGACCCUAACUUGUGAG 126GUAUUUUAGUAACAUCACAAGUCAGGCUCUUGGGACCU AGGCGGAGGGGA miR-126-1CGCUGGCGACGGGACAUUAUUACUUUUGGUACGCGCUG 127UGACACUUCAAACUCGUACCGUGAGUAAUAAUGCGCCGU CCACGGCA miR-126-2ACAUUAUUACUUUUGGUACGCGCUGUGACACUUCAAAC 128 UCGUACCGUGAGUAAUAAUGCGCmiR-127-1 UGUGAUCACUGUCUCCAGCCUGCUGAAGCUCAGAGGGCU 129CUGAUUCAGAAAGAUCAUCGGAUCCGUCUGAGCUUGGC UGGUCGGAAGUCUCAUCAUC miR-127-2CCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCAGAAAGA 130UCAUCGGAUCCGUCUGAGCUUGGCUGGUCGG miR-128aUGAGCUGUUGGAUUCGGGGCCGUAGCACUGUCUGAGAG 131GUUUACAUUUCUCACAGUGAACCGGUCUCUUUUUCAGCU GCUUC miR-128bGCCCGGCAGCCACUGUGCAGUGGGAAGGGGGGCCGAUAC 132ACUGUACGAGAGUGAGUAGCAGGUCUCACAGUGAACCG GUCUCUUUCCCUACUGUGUCACACUCCUAAUGGmiR-128 GUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGGUUUAC 133AUUUCUCACAGUGAACCGGUCUCUUUUUCAGC miR-129-1UGGAUCUUUUUGCGGUCUGGGCUUGCUGUUCCUCUCAAC 134AGUAGUCAGGAAGCCCUUACCCCAAAAAGUAUCUA MIR-129-2UGCCCUUCGCGAAUCUUUUUGCGGUCUGGGCUUGCUGUA 135CAUAACUCAAUAGCCGGAAGCCCUUACCCCAAAAAGCAU UUGCGGAGGGCG miR-130aUGCUGCUGGCCAGAGCUCUUUUCACAUUGUGCUACUGUC 136UGCACCUGUCACUAGCAGUGCAAUGUUAAAAGGGCAUU GGCCGUGUAGUG miR-131-1GCCAGGAGGCGGGGUUGGUUGUUAUCUUUGGUUAUCUA 137GCUGUAUGAGUGGUGUGGAGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAUAACCCCAUACACUGCGCAG miR-131-3CACGGCGCGGCAGCGGCACUGGCUAAGGGAGGCCCGUUU 138CUCUCUUUGGUUAUCUAGCUGUAUGAGUGCCACAGAGCC GUCAUAAAGCUAGAUAACCGAAAGUAGAAAUGmiR-131 GUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGUAUUG 139GUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAC miR-132-1CCGCCCCCGCGUCUCCAGGGCAACCGUGGCUUUCGAUUG 140UUACUGUGGGAACUGGAGGUAACAGUCUACAGCCAUGG UCGCCCCGCAGCACGCCCACGCGCmiR-132-2 GGGCAACCGUGGCUUUCGAUUGUUACUGUGGGAACUGG 141AGGUAACAGUCUACAGCCAUGGUCGCCC miR-133a-1ACAAUGCUUUGCUAGAGCUGGUAAAAUGGAACCAAAUC 142GCCUCUUCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAG CUAUGCAUUGA miR-133a-2GGGAGCCAAAUGCUUUGCUAGAGCUGGUAAAAUGGAAC 143CAAAUCGACUGUCCAAUGGAUUUGGUCCCCUUCAACCAG CUGUAGCUGUGCAUUGAUGGCGCCGmiR-133 GCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUCUUCAA 144UGGAUUUGGUCCCCUUCAACCAGCUGUAGC miR-133bCCUCAGAAGAAAGAUGCCCCCUGCUCUGGCUGGUCAAAC 145GGAACCAAGUCCGUCUUCCUGAGAGGUUUGGUCCCCUUCAACCAGCUACAGCAGGGCUGGCAAUGCCCAGUCCUUGGA GA MIR-133B-GCCCCCUGCUCUGGCUGGUCAAACGGAACCAAGUCCGUC 146 SMALLUUCCUGAGAGGUUUGGUCCCCUUCAACCAGCUACAGCAG GG miR-134-1CAGGGUGUGUGACUGGUUGACCAGAGGGGCAUGCACUG 147UGUUCACCCUGUGGGCCACCUAGUCACCAACCCUC miR-134-2AGGGUGUGUGACUGGUUGACCAGAGGGGCAUGCACUGU 148GUUCACCCUGUGGGCCACCUAGUCACCAACCCU miR-135a-1AGGCCUCGCUGUUCUCUAUGGCUUUUUAUUCCUAUGUG 149AUUCUACUGCUCACUCAUAUAGGGAUUGGAGCCGUGGC GCACGGCGGGGACA miR-135a-2AGAUAAAUUCACUCUAGUGCUUUAUGGCUUUUUAUUCC 150 (miR-135-2)UAUGUGAUAGUAAUAAAGUCUCAUGUAGGGAUGGAAGC CAUGAAAUACAUUGUGAAAAAUCA miR-135CUAUGGCUUUUUAUUCCUAUGUGAUUCUACUGCUCACUC 151 AUAUAGGGAUUGGAGCCGUGGmiR-135b CACUCUGCUGUGGCCUAUGGCUUUUCAUUCCUAUGUGAU 152UGCUGUCCCAAACUCAUGUAGGGCUAAAAGCCAUGGGCU ACAGUGAGGGGCGAGCUCC miR-136-1UGAGCCCUCGGAGGACUCCAUUUGUUUUGAUGAUGGAU 153UCUUAUGCUCCAUCAUCGUCUCAAAUGAGUCUUCAGAGG GUUCU miR-136-2GAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUAUGCUC 154 CAUCAUCGUCUCAAAUGAGUCUUCmiR-137 CUUCGGUGACGGGUAUUCUUGGGUGGAUAAUACGGAUU 155ACGUUGUUAUUGCUUAAGAAUACGCGUAGUCGAGG miR-138-1CCCUGGCAUGGUGUGGUGGGGCAGCUGGUGUUGUGAAU 156CAGGCCGUUGCCAAUCAGAGAACGGCUACUUCACAACAC CAGGGCCACACCACACUACAGG miR-138-2CGUUGCUGCAGCUGGUGUUGUGAAUCAGGCCGACGAGC 157AGCGCAUCCUCUUACCCGGCUAUUUCACGACACCAGGGU UGCAUCA miR-138CAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGCGCAUCC 158UCUUACCCGGCUAUUUCACGACACCAGGGUUG miR-139GUGUAUUCUACAGUGCACGUGUCUCCAGUGUGGCUCGG 159AGGCUGGAGACGCGGCCCUGUUGGAGUAAC miR-140UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUACCCUAU 160GGUAGGUUACGUCAUGCUGUUCUACCACAGGGUAGAAC CACGGACAGGAUACCGGGGCACC miR-140asUCCUGCCAGUGGUUUUACCCUAUGGUAGGUUACGUCAU 161GCUGUUCUACCACAGGGUAGAACCACGGACAGGA miR-140sCCUGCCAGUGGUUUUACCCUAUGGUAGGUUACGUCAUGC 162UGUUCUACCACAGGGUAGAACCACGGACAGG miR-141-1CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUGUUGGAU 163GGUCUAAUUGUGAAGCUCCUAACACUGUCUGGUAAAGA UGGCUCCCGGGUGGGUUC miR-141-2GGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCUAAUUG 164UGAAGCUCCUAACACUGUCUGGUAAAGAUGGCCC miR-142ACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGG 165 GUGUAGUGUUUCCUACUUUAUGGAUGmiR-143-1 GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGUGCUGC 166AUCUCUGGUCAGUUGGGAGUCUGAGAUGAAGCACUGUA GCUCAGGAAGAGAGAAGUUGUUCUGCAGCmiR-143-2 CCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUGGGAGU 167CUGAGAUGAAGCACUGUAGCUCAGG miR-144-1UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGUAAGUU 168UGCGAUGAGACACUACAGUAUAGAUGAUGUACUAGUCC GGGCACCCCC miR-144-2GGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCGAUGAG 169 ACACUACAGUAUAGAUGAUGUACUAGUCmiR-145-1 CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAAUCCCUU 170AGAUGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCUUG AGGUCAUGGUU miR-145-2CUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGAUGCUAA 171GAUGGGGAUUCCUGGAAAUACUGUUCUUGAG miR-146-1CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUUCCAUG 172GGUUGUGUCAGUGUCAGACCUCUGAAAUUCAGUUCUUC AGCUGGGAUAUCUCUGUCAUCGU miR-146-2AGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCAGUGUC 173 AGACCUGUGAAAUUCAGUUCUUCAGCUmiR-147 AAUCUAAAGACAACAUUUCUGCACACACACCAGACUAUG 174GAAGCCAGUGUGUGGAAAUGCUUCUGCUAGAUU miR-148aGAGGCAAAGUUCUGAGACACUCCGACUCUGAGUAUGAU 175 (miR-148)AGAAGUCAGUGCACUACAGAACUUUGUCUC miR-148bCAAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUUAUAC 176ACUCAGGCUGUGGCUCUCUGAAAGUCAGUGCAUCACAGA ACUUUGUCUCGAAAGCUUUCUA MIR-148B-AAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUUAUACA 177 SMALLCUCAGGCUGUGGCUCUCUGAAAGUCAGUGCAU miR-149-1GCCGGCGCCCGAGCUCUGGCUCCGUGUCUUCACUCCCGU 178GCUUGUCCGAGGAGGGAGGGAGGGACGGGGGCUGUGCU GGGGCAGCUGGA miR-149-2GCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUCCGAGG 179 AGGGAGGGAGGGAC miR-150-1CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCU 180GGGCUCAGACCCUGGUACAGGCCUGGGGGACAGGGACCU GGGGAC miR-150-2CCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCAGAC 181 CCUGGUACAGGCCUGGGGGACAGGGmiR-151 UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAUGUCUCA 182UCCCCUACUAGACUGAAGCUCCUUGAGGACAGG MIR-151-2CCUGUCCUCAAGGAGCUUCAGUCUAGUAGGGGAUGAGA 183CAUACUAGACUGUGAGCUCCUCGAGGGCAGG miR-152-1UGUCCCCCCCGGCCCAGGUUCUGUGAUACACUCCGACUC 184GGGCUCUGGAGCAGUCAGUGCAUGACAGAACUUGGGCCC GGAAGGACC miR-152-2GGCCCAGGUUCUGUGAUACACUCCGACUCGGGCUCUGGA 185GCAGUCAGUGCAUGACAGAACUUGGGCCCCGG miR-153-1-1CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGCAGCUAG 186UAUUCUCACUCCAGUUGCAUAGUCACAAAAGUGAUCAU UGGCAGGUGUGGC miR-153-1-2UCUCUCUCUCCCUCACAGCUGCCAGUGUCAUUGUCACAA 187AAGUGAUCAUUGGCAGGUGUGGCUGCUGCAUG miR-153-2-1AGCGGUGGCCAGUGUCAUUUUUGUGAUGUUGCAGCUAG 188UAAUAUGAGCCCAGUUGCAUAGUCACAAAAGUGAUCAU UGGAAACUGUG miR-153-2-2CAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAUAUGAG 189CCCAGUUGCAUAGUCACAAAAGUGAUCAUUG miR-154-1GUGGUACUUGAAGAUAGGUUAUCCGUGUUGCCUUCGCU 190UUAUUUGUGACGAAUCAUACACGGUUGACCUAUUUUUC AGUACCAA miR-154-2GAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAUUUGUG 191 ACGAAUCAUACACGGUUGACCUAUUUUUmiR-155 CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCCUCCAA 192CUGACUCCUACAUAUUAGCAUUAACAG MIR-156 =CCUAACACUGUCUGGUAAAGAUGGCUCCCGGGUGGGUUC 193 MIR-157 =UCUCGGCAGUAACCUUCAGGGAGCCCUGAAGACCAUGGA OVERLAP GGAC MIR-141 MIR-158-GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGA 194 SMALL =CAGCCAGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCA MIR-192UAGGUCACAGGUAUGUUCGCCUCAAUGCCAGC MIR-159-1-UCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAGUGCCC 195 SMALLACUGGUUCCAGUGGGGCUGCUGUUAUCUGGGGCGAGGG CCA MIR-161-AAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGGUGACU 196 SMALLGGUCUGGGCUACGCUAUGCUGCGGCGCUCGGG MIR-163-1B-CAUUGGCCUCCUAAGCCAGGGAUUGUGGGUUCGAGUCCC 197 SMALLACCCGGGGUAAAGAAAGGCCGAAUU MIR-163-3-CCUAAGCCAGGGAUUGUGGGUUCGAGUCCCACCUGGGGU 198 SMALLAGAGGUGAAAGUUCCUUUUACGGAAUUUUUU miR-162CAAUGUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGC 199GUUAAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUGCU GCUGAAGUAAGGUUGACCAUACUCUACAGUUGMIR-175- GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUAGAUGA 200 SMALL =UUGUGCAUUGUUUCAAAAUGGUGCCCUAGUGACUACAA MIR-224 AGCCC MIR-177-ACGCAAGUGUCCUAAGGUGAGCUCAGGGAGCACAGAAA 201 SMALLCCUCCAGUGGAACAGAAGGGCAAAAGCUCAUU MIR-180-CAUGUGUCACUUUCAGGUGGAGUUUCAAGAGUCCCUUCC 202 SMALLUGGUUCACCGUCUCCUUUGCUCUUCCACAAC miR-181aAGAAGGGCUAUCAGGCCAGCCUUCAGAGGACUCCAAGGA 203ACAUUCAACGCUGUCGGUGAGUUUGGGAUUUGAAAAAA CCACUGACCGUUGACUGUACCUUGGGGUCCUUAmiR-181b-1 CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAAUCAAC 204AUUCAUUGCUGUCGGUGGGUUGAACUGUGUGGACAAGCUCACUGAACAAUGAAUGCAACUGUGGCCCCGCUU miR-181b-2CUGAUGGCUGCACUCAACAUUCAUUGCUGUCGGUGGGU 205UUGAGUCUGAAUCAACUCACUGAUCAAUGAAUGCAAAC UGCGGACCAAACA miR-181cCGGAAAAUUUGCCAAGGGUUUGGGGGAACAUUCAACCU 206GUCGGUGAGUUUGGGCAGCUCAGGCAAACCAUCGACCGUUGAGUGGACCCUGAGGCCUGGAAUUGCCAUCCU miR-182-asGAGCUGCUUGCCUCCCCCCGUUUUUGGCAAUGGUAGAAC 207UCACACUGGUGAGGUAACAGGAUCCGGUGGUUCUAGAC UUGCCAACUAUGGGGCGAGGACUCAGCCGGCACmiR-182 UUUUUGGCAAUGGUAGAACUCACACUGGUGAGGUAACA 208GGAUCCGGUGGUUCUAGACUUGCCAACUAUGG miR-183CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCACUGGU 209AGAAUUCACUGUGAACAGUCUCAGUCAGUGAAUUACCGAAGGGCCAUAAACAGAGCAGAGACAGAUCCACGA miR-184-1CCAGUCACGUCCCCUUAUCACUUUUCCAGCCCAGCUUUG 210UGACUGUAAGUGUUGGACGGAGAACUGAUAAGGGUAGG UGAUUGA miR-184-2CCUUAUCACUUUUCCAGCCCAGCUUUGUGACUGUAAGUG 211 UUGGACGGAGAACUGAUAAGGGUAGGmiR-185-1 AGGGGGCGAGGGAUUGGAGAGAAAGGCAGUUCCUGAUG 212GUCCCCUCCCCAGGGGCUGGCUUUCCUCUGGUCCUUCCC UCCCA miR-185-2AGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUCCCCUC 213 CCCAGGGGCUGGCUUUCCUCUGGUCCUUmiR-186-1 UGCUUGUAACUUUCCAAAGAAUUCUCCUUUUGGGCUUU 214CUGGUUUUAUUUUAAGCCCAAAGGUGAAUUUUUUGGGA AGUUUGAGCU miR-186-2ACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUGGUUUU 215AUUUUAAGCCCAAAGGUGAAUUUUUUGGGAAGU miR-187GGUCGGGCUCACCAUGACACAGUGUGAGACUCGGGCUAC 216AACACAGGACCCGGGGCGCUGCUCUGACCCCUCGUGUCU UGUGUUGCAGCCGGAGGGACGCAGGUCCGCAmiR-188-1 UGCUCCCUCUCUCACAUCCCUUGCAUGGUGGAGGGUGAG 217CUUUCUGAAAACCCCUCCCACAUGCAGGGUUUGCAGGAU GGCGAGCC miR-188-2UCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUUUCUGA 218AAACCCCUCCCACAUGCAGGGUUUGCAGGA miR-189-1CUGUCGAUUGGACCCGCCCUCCGGUGCCUACUGAGCUGA 219UAUCAGUUCUCAUUUUACACACUGGCUCAGUUCAGCAGG AACAGGAGUCGAGCCCUUGAGCAAmiR-189-2 CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUAC 220ACACUGGCUCAGUUCAGCAGGAACAGGAG miR-190-1UGCAGGCCUCUGUGUGAUAUGUUUGAUAUAUUAGGUUG 221UUAUUUAAUCCAACUAUAUAUCAAACAUAUUCCUACAG UGUCUUGCC miR-190-2CUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAUUUAAU 222 CCAACUAUAUAUCAAACAUAUUCCUACAGmiR-191-1 CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGCAGCUGU 223UGUCUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCC CUGCUCUCCUGCCU miR-191-2AGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAG 224AGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCU miR-192-2/3CCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGAC 225AGCCAGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAU AGGUCACAGGUAUGUUCGCCUCAAUGCCAGmiR-192 GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGA 226CAGCCAGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCA UAGGUCACAGGUAUGUUCGCCUCAAUGCCAGCmiR-193-1 CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGGGCGAG 227AUGAGGGUGUCGGAUCAACUGGCCUACAAAGUCCCAGU UCUCGGCCCCCG miR-193-2GCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGGAUCAA 228 CUGGCCUACAAAGUCCCAGUmiR-194-1 AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGUGGACU 229GUGUACCAAUUUCCAGUGGAGAUGCUGUUACUUUUGAU GGUUACCAA miR-194-2GUGUAACAGCAACUCCAUGUGGACUGUGUACCAAUUUCC 230 AGUGGAGAUGCUGUUACUUUUGAUmiR-195-1 AGCUUCCCUGGCUCUAGCAGCACAGAAAUAUUGGCACAG 231GGAAGCGAGUCUGCCAAUAUUGGCUGUGCUGCUCCAGGC AGGGUGGUG miR-195-2UAGCAGCACAGAAAUAUUGGCACAGGGAAGCGAGUCUG 232 CCAAUAUUGGCUGUGCUGCUmiR-196-1 CUAGAGCUUGAAUUGGAACUGCUGAGUGAAUUAGGUAG 233UUUCAUGUUGUUGGGCCUGGGUUUCUGAACACAACAACAUUAAACCACCCGAUUCACGGCAGUUACUGCUCC miR-196a-1GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUC 234UGAACACAACAACAUUAAACCACCCGAUUCAC miR-196a-2UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUUUCAUG 235 (miR-196-2)UUGUUGGGAUUGAGUUUUGAACUCGGCAACAAGAAACUGCCUGAGUUACAUCAGUCGGUUUUCGUCGAGGGC miR-196GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUC 236UGAACACAACAACAUUAAACCACCCGAUUCAC miR-196bACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUUGGGAU 237CCACCUUUCUCUCGACAGCACGACACUGCCUUCAUUACU UCAGUUG miR-197GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUAAGAGC 238UCUUCACCCUUCACCACCUUCUCCACCCAGCAUGGCC MIR-197-2GUGCAUGUGUAUGUAUGUGUGCAUGUGCAUGUGUAUGU 239 GUAUGAGUGCAUGCGUGUGUGCmiR-198 UCAUUGGUCCAGAGGGGAGAUAGGUUCCUGUGAUUUUU 240CCUUCUUCUCUAUAGAAUAAAUGA miR-199a-1GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUC 241AAUGUGUACAGUAGUCUGCACAUUGGUUAGGC miR-199a-2AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUU 242CAGACUACCUGUUCAGGACAAUGCCGUUGUACAGUAGUC UGCACAUUGGUUAGACUGGGCAAGGGAGAGCAmiR-199b CCAGAGGACACCUCCACUCCGUCUACCCAGUGUUUAGAC 243UAUCUGUUCAGGACUCCCAAAUUGUACAGUAGUCUGCAC AUUGGUUAGGCUGGGCUGGGUUAGACCCUCGGmiR-199s GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUC 244AAUGUGUACAGUAGUCUGCACAUUGGUUAGGC miR-200aGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGGAGUCA 245GGUCUCUAAUACUGCCUGGUAAUGAUGACGGC miR-200bCCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGCAGCAUU 246GGAUGGAGUCAGGUCUCUAAUACUGCCUGGUAAUGAUG ACGGCGGAGCCCUGCACG miR-200cCCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGUUGGGAG 247UCUCUAAUACUGCCGGGUAAUGAUGGAGG miR-202GUUCCUUUUUCCUAUGCAUAUACUUCUUUGAGGAUCUG 248GCCUAAAGAGGUAUAGGGCAUGGGAAGAUGGAGC miR-203GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUUCUUAA 249CAGUUCAACAGUUCUGUAGCGCAAUUGUGAAAUGUUUAGGACCACUAGACCCGGCGGGCGCGGCGACAGCGA miR-204GGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUU 250UGUCAUCCUAUGCCUGAGAAUAUAUGAAGGAGGCUGGG AAGGCAAAGGGACGUUCAAUUGUCAUCACUGGCmiR-205 AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUC 251AUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAG UGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAmiR-206-1 UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCCAUAUG 252GAUUACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGUUU CGGCAAGUG miR-206-2AGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUACUUU 253GCUAUGGAAUGUAAGGAAGUGUGUGGUUUU miR-208UGACGGGCGAGCUUUUGGCCCGGGUUAUACCUGAUGCUC 254ACGUAUAAGACGAGCAAAAAGCUUGUUGGUCA miR-210ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCUGCCCA 255CCGCACACUGCGCUGCCCCAGACCCACUGUGCGUGUGAC AGCGGCUGAUCUGUGCCUGGGCAGCGCGACCCmiR-211 UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUUGUCAUC 256CUUCGCCUAGGGCUCUGAGCAGGGCAGGGACAGCAAAGG GGUGCUCAGUUGUCACUUCCCACAGCACGGAGmiR-212 CGGGGCACCCCGCCCGGACAGCGCGCCGGCACCUUGGCU 257CUAGACUGCUUACUGCCCGGGCCGCCCUCAGUAACAGUC UCCAGUCACGGCCACCGACGCCUGGCCCCGCCmiR-213-2 CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAAUCAAC 258AUUCAUUGCUGUCGGUGGGUUGAACUGUGUGGACAAGCUCACUGAACAAUGAAUGCAACUGUGGCCCCGCUU miR-213GAGUUUUGAGGUUGCUUCAGUGAACAUUCAACGCUGUC 259GGUGAGUUUGGAAUUAAAAUCAAAACCAUCGACCGUUG AUUGUACCCUAUGGCUAACCAUCAUCUACUCCmiR-214 GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCCUGUCU 260ACACUUGCUGUGCAGAACAUCCGCUCACCUGUACAGCAGGCACAGACAGGCAGUCACAUGACAACCCAGCCU miR-215AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCUAUGAA 261UUGACAGACAAUAUAGCUGAGUUUGUCUGUCAUUUCUUUAGGCCAAUAUUCUGUAUGACUGUGCUACUUCAA miR-216GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCAACUGU 262GAGAUGUUCAUACAAUCCCUCACAGUGGUCUCUGGGAUUAUGCUAAACAGAGCAAUUUCCUAGCCCUCACGA miR-217AGUAUAAUUAUUACAUAGUUUUUGAUGUCGCAGAUACU 263GCAUCAGGAACUGAUUGGAUAAGAAUCAGUCACCAUCAGUUCCUAAUGCAUUGCCUUCAGCAUCUAAACAAG miR-218-1GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUGAUCUA 264ACCAUGUGGUUGCGAGGUAUGAGUAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA miR-218-2GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUGAUCUAA 265CCAUGUGGUGGAACGAUGGAAACGGAACAUGGUUCUGU CAAGCACCGCGGAAAGCACCGUGCUCUCCUGCAmiR-219 CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACGCAAUU 266CUCGAGUCUAUGGCUCCGGCCGAGAGUUGAGUCUGGACG UCCCGAGCCGCCGCCCCCAAACCUCGAGCGGGmiR-219-1 CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACGCAAUU 267CUCGAGUCUAUGGCUCCGGCCGAGAGUUGAGUCUGGACG UCCCGAGCCGCCGCCCCCAAACCUCGAGCGGGmiR-219-2 ACUCAGGGGCUUCGCCACUGAUUGUCCAAACGCAAUUCU 268UGUACGAGUCUGCGGCCAACCGAGAAUUGUGGCUGGAC AUCUGUGGCUGAGCUCCGGG miR-220GACAGUGUGGCAUUGUAGGGCUCCACACCGUAUCUGACA 269CUUUGGGCGAGGGCACCAUGCUGAAGGUGUUCAUGAUG CGGUCUGGGAACUCCUCACGGAUCUUACUGAUGmiR-221 UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAU 270GUAGAUUUCUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCAGGCUACCUGGAAACAUGUUCUC miR-222GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUCAGUAG 271CCAGUGUAGAUCCUGUCUUUCGUAAUCAGCAGCUACAUCUGGCUACUGGGUCUCUGAUGGCAUCUUCUAGCU miR-223CCUGGCCUCCUGCAGUGCCACGCUCCGUGUAUUUGACAA 272GCUGAGUUGGACACUCCAUGUGGUAGAGUGUCAGUUUG UCAAAUACCCCAAGUGCGGCACAUGCUUACCAGmiR-224 GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUAGAUGA 273UUGUGCAUUGUUUCAAAAUGGUGCCCUAGUGACUACAA AGCCC MIR-294-1CAAUCUUCCUUUAUCAUGGUAUUGAUUUUUCAGUGCUU 274 (CHR16)CCCUUUUGUGUGAGAGAAGAUA miR-296 AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUGUGC275 CUAAUUCAGAGGGUUGGGUGGAGGCUCUCCUGAAGGGC UCU miR-299AAGAAAUGGUUUACCGUCCCACAUACAUUUUGAAUAUG 276 UAUGUGGGAUGGUAAACCGCUUCUUmiR-301 ACUGCUAACGAAUGCUCUGACUUUAUUGCACUACUGUAC 277UUUACAGCUAGCAGUGCAAUAGUAUUGUCAAAGCAUCU GAAAGCAGG miR-302aCCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAA 278AGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG miR-302bGCUCCCUUCAACUUUAACAUGGAAGUGCUUUCUGUGACU 279UUAAAAGUAAGUGCUUCCAUGUUUUAGUAGGAGU miR-302cCCUUUGCUUUAACAUGGGGGUACCUGCUGUGUGAAACA 280AAAGUAAGUGCUUCCAUGUUUCAGUGGAGG miR-302dCCUCUACUUUAACAUGGAGGCACUUGCUGUGACAUGACA 281AAAAUAAGUGCUUCCAUGUUUGAGUGUGG miR-320GCUUCGCUCCCCUCCGCCUUCUCUUCCCGGUUCUUCCCG 282GAGUCGGGAAAAGCUGGGUUGAGAGGGCGAAAAAGGAU GAGGU miR-321UUGGCCUCCUAAGCCAGGGAUUGUGGGUUCGAGUCCCAC 283 CCGGGGUAAAGAAAGGCCGA miR-323UUGGUACUUGGAGAGAGGUGGUCCGUGGCGCGUUCGCU 284UUAUUUAUGGCGCACAUUACACGGUCGACCUCUUUGCAG UAUCUAAUC miR-324CUGACUAUGCCUCCCCGCAUCCCCUAGGGCAUUGGUGUA 285AAGCUGGAGACCCACUGCCCCAGGUGCUGCUGGGGGUUG UAGUC miR-325AUACAGUGCUUGGUUCCUAGUAGGUGUCCAGUAAGUGU 286UUGUGACAUAAUUUGUUUAUUGAGGACCUCCUAUCAAU CAAGCACUGUGCUAGGCUCUGG miR-326CUCAUCUGUCUGUUGGGCUGGAGGCAGGGCCUUUGUGA 287AGGCGGGUGGUGCUCAGAUCGCCUCUGGGCCCUUCCUCC AGCCCCGAGGCGGAUUCA miR-328UGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAAAGUGC 288AUACAGCCCCUGGCCCUCUCUGCCCUUCCGUCCCCUG miR-330CUUUGGCGAUCACUGCCUCUCUGGGCCUGUGUCUUAGGC 289UCUGCAAGAUCAACCGAGCAAAGCACACGGCCUGCAGAG AGGCAGCGCUCUGCCC miR-331GAGUUUGGUUUUGUUUGGGUUUGUUCUAGGUAUGGUCC 290CAGGGAUCCCAGAUCAAACCAGGCCCCUGGGCCUAUCCU AGAACCAACCUAAGCUC miR-335UGUUUUGAGCGGGGGUCAAGAGCAAUAACGAAAAAUGU 291UUGUCAUAAACCGUUUUUCAUUAUUGCUCCUGACCUCCU CUCAUUUGCUAUAUUCA miR-337GUAGUCAGUAGUUGGGGGGUGGGAACGGCUUCAUACAG 292GAGUUGAUGCACAGUUAUCCAGCUCCUAUAUGAUGCCU UUCUUCAUCCCCUUCAA miR-338UCUCCAACAAUAUCCUGGUGCUGAGUGAUGACUCAGGCG 293 ACUCCAGCAUCAGUGAUUUUGUUGAAGAmiR-339 CGGGGCGGCCGCUCUCCCUGUCCUCCAGGAGCUCACGUG 294UGCCUGCCUGUGAGCGCCUCGACGACAGAGCCGGCGCCU GCCCCAGUGUCUGCGC miR-340UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACUGAUUG 295UCAUAUGUCGUUUGUGGGAUCCGUCUCAGUUACUUUAU AGCCAUACCUGGUAUCUUA miR-342GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGUGAUUG 296AGGGACAUGGUUAAUGGAAUUGUCUCACACAGAAAUCG CACCCGUCACCUUGGCCUACUUA miR-345ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAGGGCUC 297GUGAUGGCUGGUGGGCCCUGAACGAGGGGUCUGGAGGC CUGGGUUUGAAUAUCGACAGC miR-346GUCUGUCUGCCCGCAUGCCUGCCUCUCUGUUGCUCUGAA 298GGAGGCAGGGGCUGGGCCUGCAGCUGCCUGGGCAGAGCG GCUCCUGC miR-367CCAUUACUGUUGCUAAUAUGCAACUCUGUUGAAUAUAA 299AUUGGAAUUGCACUUUAGCAAUGGUGAUGG miR-368AAAAGGUGGAUAUUCCUUCUAUGUUUAUGUUAUUUAUG 300 GUUAAACAUAGAGGAAAUUCCACGUUUUmiR-369 UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUUAUUGA 301CUUCGAAUAAUACAUGGUUGAUCUUUUCUCAG miR-370AGACAGAGAAGCCAGGUCACGUCUCUGCAGUUACACAGC 302UCACGAGUGCCUGCUGGGGUGGAACCUGGUCUGUCU miR-371GUGGCACUCAAACUGUGGGGGCACUUUCUGCUCUCUGGU 303 GAAAGUGCCGCCAUCUUUUGAGUGUUACmiR-372 GUGGGCCUCAAAUGUGGAGCACUAUUCUGAUGUCCAAG 304UGGAAAGUGCUGCGACAUUUGAGCGUCAC miR-373GGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUGUCUGU 305ACUGGGAAGUGCUUCGAUUUUGGGGUGUCCC miR-374UACAUCGGCCAUUAUAAUACAACCUGAUAAGUGUUAUA 306GCACUUAUCAGAUUGUAUUGUAAUUGUCUGUGUA mir-hes1AUGGAGCUGCUCACCCUGUGGGCCUCAAAUGUGGAGGA 307ACUAUUCUGAUGUCCAAGUGGAAAGUGCUGCGACAUUU GAGCGUCACCGGUGACGCCCAUAUCAmir-hes2 GCAUCCCCUCAGCCUGUGGCACUCAAACUGUGGGGGCAC 308UUUCUGCUCUCUGGUGAAAGUGCCGCCAUCUUUUGAGU GUUACCGCUUGAGAAGACUCAACC mir-hes3CGAGGAGCUCAUACUGGGAUACUCAAAAUGGGGGCGCU 309UUCCUUUUUGUCUGUUACUGGGAAGUGCUUCGAUUUUG GGGUGUCCCUGUUUGAGUAGGGCAUC *Anunderlined sequence within a precursor sequence corresponds to a matureprocessed miR transcript (see Table 1b). Some precursor sequences havetwo underlined sequences denoting two different mature miRs that arederived from the same precursor. All sequences are human.

TABLE 1b Human Mature microRNA Sequences. Mature miRNAMature miRNA Sequence SEQ ID Corresponding precursor Name (5′ to 3′) NO.microRNA(s); see Table 1a let-7a UGAGGUAGUAGGUUGUA 310let-7a-1; let-7a-2; let-7a-3; UAGUU let-7a-4 let-7b UGAGGUAGUAGGUUGUG311 let-7b UGGUU let-7c UGAGGUAGUAGGUUGUA 312 let-7c UGGUU let-7dAGAGGUAGUAGGUUGCA 313 let-7d; let-7d-v1 UAGU let-7e UGAGGUAGGAGGUUGUA314 let-7e UAGU let-7f UGAGGUAGUAGAUUGUA 315 let-7f-1; let-7f-2-1; UAGUUlet-7f-2-2 let-7g UGAGGUAGUAGUUUGUA 316 let-7g CAGU let-7iUGAGGUAGUAGUUUGUG 317 let-7i CU miR-1 UGGAAUGUAAAGAAGUA 318miR-1b; miR-1b-1; UGUA miR-1b-2 miR-7 UGGAAGACUAGUGAUUU 319miR-7-1; miR-7-1a; UGUU miR-7-2; miR-7-3 miR-9 UCUUUGGUUAUCUAGCU 320miR-9-1; miR-9-2; GUAUGA miR-9-3 miR-9* UAAAGCUAGAUAACCGA 321miR-9-1; miR-9-2; AAGU miR-9-3 miR-10a UACCCUGUAGAUCCGAA 322 miR-10aUUUGUG miR-10b UACCCUGUAGAACCGAA 323 miR-10b UUUGU miR-15aUAGCAGCACAUAAUGGU 324 miR-15a; miR-15a-2 UUGUG miR-15b UAGCAGCACAUCAUGGU325 miR-15b UUACA miR-16 UAGCAGCACGUAAAUAU 326 miR-16-1; miR-16-2; UGGCGmiR-16-13 miR-17-5p CAAAGUGCUUACAGUGC 327 miR-17 AGGUAGU miR-17-3pACUGCAGUGAAGGCACU 328 miR-17 UGU miR-18 UAAGGUGCAUCUAGUGC 329miR-18; miR-18-13 AGAUA miR-19a UGUGCAAAUCUAUGCAA 330miR-19a; miR-19a-13 AACUGA miR-19b UGUGCAAAUCCAUGCAA 331miR-19b-1; miR-19b-2 AACUGA miR-20 UAAAGUGCUUAUAGUGC 332miR-20 (miR-20a) AGGUA miR-21 UAGCUUAUCAGACUGAU 333 miR-21; miR-21-17GUUGA miR-22 AAGCUGCCAGUUGAAGA 334 miR-22 ACUGU miR-23aAUCACAUUGCCAGGGAU 335 miR-23a UUCC miR-23b AUCACAUUGCCAGGGAU 336 miR-23bUACCAC miR-24 UGGCUCAGUUCAGCAGG 337 miR-24-1; miR-24-2; AACAGmiR-24-19; miR-24-9 miR-25 CAUUGCACUUGUCUCGG 338 miR-25 UCUGA miR-26aUUCAAGUAAUCCAGGAU 339 miR-26a; miR-26a-1; AGGCU miR-26a-2 miR-26bUUCAAGUAAUUCAGGAU 340 miR-26b AGGU miR-27a UUCACAGUGGCUAAGUU 341 miR-27aCCGCC miR-27b UUCACAGUGGCUAAGUU 342 miR-27b-1; miR-27b-2 CUG miR-28AAGGAGCUCACAGUCUA 343 miR-28 UUGAG miR-29a CUAGCACCAUCUGAAAU 344miR-29a-2; miR-29a CGGUU miR-29b UAGCACCAUUUGAAAUC 345miR-29b-1; miR-29b-2 AGU miR-29c UAGCACCAUUUGAAAUC 346 miR-29c GGUUAmiR-30a-5p UGUAAACAUCCUCGACU 347 miR-30a GGAAGC miR-30a-3pCUUUCAGUCGGAUGUUU 348 miR-30a GCAGC miR-30b UGUAAACAUCCUACACU 349miR-30b-1; miR-30b-2 CAGC miR-30c UGUAAACAUCCUACACU 350 miR-30c CUCAGCmiR-30d UGUAAACAUCCCCGACU 351 miR-30d GGAAG miR-30e UGUAAACAUCCUUGACU352 miR-30e GGA miR-31 GGCAAGAUGCUGGCAUA 353 miR-31 GCUG miR-32UAUUGCACAUUACUAAG 354 miR-32 UUGC miR-33 GUGCAUUGUAGUUGCAU 355miR-33; miR-33b UG miR-34a UGGCAGUGUCUUAGCUG 356 miR-34a GUUGU miR-34bAGGCAGUGUCAUUAGCU 357 miR-34b GAUUG miR-34c AGGCAGUGUAGUUAGCU 358miR-34c GAUUG miR-92 UAUUGCACUUGUCCCGG 359 miR-92-2; miR-92-1 CCUGUmiR-93 AAAGUGCUGUUCGUGCA 360 miR-93-1; miR-93-2 GGUAG miR-95UUCAACGGGUAUUUAUU 361 miR-95 GAGCA miR-96 UUUGGCACUAGCACAUU 362 miR-96UUUGC miR-98 UGAGGUAGUAAGUUGUA 363 miR-98 UUGUU miR-99aAACCCGUAGAUCCGAUC 364 miR-99a UUGUG miR-99b CACCCGUAGAACCGACC 365miR-99b UUGCG miR-100 UACAGUACUGUGAUAAC 366 miR-100 UGAAG miR-101UACAGUACUGUGAUAAC 367 miR-101-1; miR-101-2 UGAAG miR-103AGCAGCAUUGUACAGGG 368 miR-103-1 CUAUGA miR-105 UCAAAUGCUCAGACUCC 369miR-105 UGU miR-106-a AAAAGUGCUUACAGUGC 370 miR-106-a AGGUAGC miR-106-bUAAAGUGCUGACAGUGC 371 miR-106-b AGAU miR-107 AGCAGCAUUGUACAGGG 372miR-107 CUAUCA miR-122a UGGAGUGUGACAAUGGU 373 miR-122a-1; miR-122a-2GUUUGU miR-124a UUAAGGCACGCGGUGAA 374 miR-124a-1; miR-124a-2; UGCCAmiR-124a-3 miR-125a UCCCUGAGACCCUUUAA 375 miR-125a-1; miR-125a-2 CCUGUGmiR-125b UCCCUGAGACCCUAACU 376 miR-125b-1; miR-125b-2 UGUGA miR-126*CAUUAUUACUUUUGGUA 377 miR-126-1; miR-126-2 CGCG miR-126UCGUACCGUGAGUAAUA 378 miR-126-1; miR-126-2 AUGC miR-127UCGGAUCCGUCUGAGCU 379 miR-127-1; miR-127-2 UGGCU miR-128aUCACAGUGAACCGGUCU 380 miR-128; miR-128a CUUUU miR-128b UCACAGUGAACCGGUCU381 miR-128b CUUUC miR-129 CUUUUUGCGGUCUGGGC 382 miR-129-1; miR-129-2UUGC miR-130a CAGUGCAAUGUUAAAAG 383 miR-130a GGC miR-130bCAGUGCAAUGAUGAAAG 384 miR-130b GGCAU miR-132 UAACAGUCUACAGCCAU 385miR-132-1 GGUCG miR-133a UUGGUCCCCUUCAACCA 386 miR-133a-1; miR-133a-2GCUGU miR-133b UUGGUCCCCUUCAACCA 387 miR-133b GCUA miR-134UGUGACUGGUUGACCAG 388 miR-134-1; miR-134-2 AGGG miR-135aUAUGGCUUUUUAUUCCU 389 miR-135a; miR-135a-2 AUGUGA (miR-135-2) miR-135bUAUGGCUUUUCAUUCCU 390 miR-135b AUGUG miR-136 ACUCCAUUUGUUUUGAU 391miR-136-1; miR-136-2 GAUGGA miR-137 UAUUGCUUAAGAAUACG 392 miR-137 CGUAGmiR-138 AGCUGGUGUUGUGAAUC 393 miR-138-1; miR-138-2 miR-139UCUACAGUGCACGUGUC 394 miR-139 U miR-140 AGUGGUUUUACCCUAUG 395miR-140; miR-140as; GUAG miR-140s miR-141 AACACUGUCUGGUAAAG 396miR-141-1; miR-141-2 AUGG miR-142-3p UGUAGUGUUUCCUACUU 397 miR-142UAUGGA miR-142-5p CAUAAAGUAGAAAGCAC 398 miR-142 UAC miR-143UGAGAUGAAGCACUGUA 399 miR-143-1 GCUCA miR-144 UACAGUAUAGAUGAUGU 400miR-144-1; miR-144-2 ACUAG miR-145 GUCCAGUUUUCCCAGGA 401miR-145-1; miR-145-2 AUCCCUU miR-146 UGAGAACUGAAUUCCAU 402miR-146-1; miR-146-2 GGGUU miR-147 GUGUGUGGAAAUGCUUC 403 miR-147 UGCmiR-148a UCAGUGCACUACAGAAC 404 miR-148a (miR-148) UUUGU miR-148bUCAGUGCAUCACAGAAC 405 miR-148b UUUGU miR-149 UCUGGCUCCGUGUCUUC 406miR-149 ACUCC miR-150 UCUCCCAACCCUUGUACC 407 miR-150-1; miR-150-2 AGUGmiR-151 ACUAGACUGAAGCUCCU 408 miR-151 UGAGG miR-152 UCAGUGCAUGACAGAAC409 miR-152-1; miR-152-2 UUGG miR-153 UUGCAUAGUCACAAAAG 410miR-153-1-1; miR-153-1-2; UGA miR-153-2-1; miR-153-2-2 miR-154UAGGUUAUCCGUGUUGC 411 miR-154-1; miR-154-2 CUUCG miR-154*AAUCAUACACGGUUGAC 412 miR-154-1; miR-154-2 CUAUU miR-155UUAAUGCUAAUCGUGAU 413 miR-155 AGGGG miR-181a AACAUUCAACGCUGUCG 414miR-181a GUGAGU miR-181b AACAUUCAUUGCUGUCG 415 miR-181b-1; miR-181b-2GUGGGUU miR-181c AACAUUCAACCUGUCGG 416 miR-181c UGAGU miR-182UUUGGCAAUGGUAGAAC 417 miR-182; miR-182as UCACA miR-182*UGGUUCUAGACUUGCCA 418 miR-182; miR-182as ACUA miR-183 UAUGGCACUGGUAGAAU419 miR-183 UCACUG miR-184 UGGACGGAGAACUGAUA 420 miR-184-1; miR-184-2AGGGU miR-185 UGGAGAGAAAGGCAGUU 421 miR-185-1; miR-185-2 C miR-186CAAAGAAUUCUCCUUUU 422 miR-186-1; miR-186-2 GGGCUU miR-187UCGUGUCUUGUGUUGCA 423 miR-187 GCCG miR-188 CAUCCCUUGCAUGGUGG 424 miR-188AGGGU miR-189 GUGCCUACUGAGCUGAU 425 miR-189-1; miR-189-2 AUCAGU miR-190UGAUAUGUUUGAUAUAU 426 miR-190-1; miR-190-2 UAGGU miR-191CAACGGAAUCCCAAAAG 427 miR-191-1; miR-191-2 CAGCU miR-192CUGACCUAUGAAUUGAC 428 miR-192 AGCC miR-193 AACUGGCCUACAAAGUC 429miR-193-1; miR-193-2 CCAG miR-194 UGUAACAGCAACUCCAU 430miR-194-1; miR-194-2 GUGGA miR-195 UAGCAGCACAGAAAUAU 431miR-195-1; miR-195-2 UGGC miR-196a UAGGUAGUUUCAUGUUG 432miR-196a; miR-196a-2 UUGG (miR196-2) miR-196b UAGGUAGUUUCCUGUUG 433miR-196b UUGG miR-197 UUCACCACCUUCUCCACC 434 miR-197 CAGC miR-198GGUCCAGAGGGGAGAUA 435 miR-198 GG miR-199a CCCAGUGUUCAGACUAC 436miR-199a-1; miR-199a-2 CUGUUC miR-199a* UACAGUAGUCUGCACAU 437miR-199a-1; miR-199a-2; UGGUU miR-199s; miR-199b miR-199bCCCAGUGUUUAGACUAU 438 miR-199b CUGUUC miR-200a UAACACUGUCUGGUAAC 439miR-200a GAUGU miR-200b CUCUAAUACUGCCUGGU 440 miR-200b AAUGAUG miR-200cAAUACUGCCGGGUAAUG 441 miR-200c AUGGA miR-202 AGAGGUAUAGGGCAUGG 442miR-202 GAAGA miR-203 GUGAAAUGUUUAGGACC 443 miR-203 ACUAG miR-204UUCCCUUUGUCAUCCUA 444 miR-204 UGCCU miR-205 UCCUUCAUUCCACCGGA 445miR-205 GUCUG miR-206 UGGAAUGUAAGGAAGUG 446 miR-206-1; miR-206-2 UGUGGmiR-208 AUAAGACGAGCAAAAAG 447 miR-208 CUUGU miR-210 CUGUGCGUGUGACAGCG448 miR-210 GCUG miR-211 UUCCCUUUGUCAUCCUU 449 miR-211 CGCCU miR-212UAACAGUCUCCAGUCAC 450 miR-212 GGCC miR-213 ACCAUCGACCGUUGAUU 451 miR-213GUACC miR-214 ACAGCAGGCACAGACAG 452 miR-214 GCAG miR-215AUGACCUAUGAAUUGAC 453 miR-215 AGAC miR-216 UAAUCUCAGCUGGCAAC 454 miR-216UGUG miR-217 UACUGCAUCAGGAACUG 455 miR-217 AUUGGAU miR-218UUGUGCUUGAUCUAACC 456 miR-218-1; miR-218-2 AUGU miR-219UGAUUGUCCAAACGCAA 457 miR-219; miR-219-1; UUCU miR-219-2 miR-220CCACACCGUAUCUGACA 458 miR-220 CUUU miR-221 AGCUACAUUGUCUGCUG 459 miR-221GGUUUC miR-222 AGCUACAUCUGGCUACU 460 miR-222 GGGUCUC miR-223UGUCAGUUUGUCAAAUA 461 miR-223 CCCC miR-224 CAAGUCACUAGUGGUUC 462 miR-224CGUUUA miR-296 AGGGCCCCCCCUCAAUCC 463 miR-296 UGU miR-299UGGUUUACCGUCCCACA 464 miR-299 UACAU miR-301 CAGUGCAAUAGUAUUGU 465miR-301 CAAAGC miR-302a UAAGUGCUUCCAUGUUU 466 miR-302a UGGUGA miR-302b*ACUUUAACAUGGAAGUG 467 miR-302b CUUUCU miR-302b UAAGUGCUUCCAUGUUU 468miR-302b UAGUAG miR-302c* UUUAACAUGGGGGUACC 469 miR-302c UGCUG miR-302cUAAGUGCUUCCAUGUUU 470 miR-302c CAGUGG miR-302d UAAGUGCUUCCAUGUUU 471miR-302d GAGUGU miR-320 AAAAGCUGGGUUGAGAG 472 miR-320 GGCGAA miR-321UAAGCCAGGGAUUGUGG 473 miR-321 GUUC miR-323 GCACAUUACACGGUCGA 474 miR-323CCUCU miR-324-5p CGCAUCCCCUAGGGCAU 475 miR-324 UGGUGU miR-324-3pCCACUGCCCCAGGUGCU 476 miR-324 GCUGG miR-325 CCUAGUAGGUGUCCAGU 477miR-325 AAGU miR-326 CCUCUGGGCCCUUCCUCC 478 miR-326 AG miR-328CUGGCCCUCUCUGCCCUU 479 miR-328 CCGU miR-330 GCAAAGCACACGGCCUG 480miR-330 CAGAGA miR-331 GCCCCUGGGCCUAUCCU 481 miR-331 AGAA miR-335UCAAGAGCAAUAACGAA 482 miR-335 AAAUGU miR-337 UCCAGCUCCUAUAUGAU 483miR-337 GCCUUU miR-338 UCCAGCAUCAGUGAUUU 484 miR-338 UGUUGA miR-339UCCCUGUCCUCCAGGAG 485 miR-339 CUCA miR-340 UCCGUCUCAGUUACUUU 486 miR-340AUAGCC miR-342 UCUCACACAGAAAUCGC 487 miR-342 ACCCGUC miR-345UGCUGACUCCUAGUCCA 488 miR-345 GGGC miR-346 UGUCUGCCCGCAUGCCU 489 miR-346GCCUCU miR-367 AAUUGCACUUUAGCAAU 490 miR-367 GGUGA miR-368ACAUAGAGGAAAUUCCA 491 miR-368 CGUUU miR-369 AAUAAUACAUGGUUGAU 492miR-369 CUUU miR-370 GCCUGCUGGGGUGGAAC 493 miR-370 CUGG miR-371GUGCCGCCAUCUUUUGA 494 miR-371 GUGU miR-372 AAAGUGCUGCGACAUUU 495 miR-372GAGCGU miR-373* ACUCAAAAUGGGGGCGC 496 miR-373 UUUCC miR-373GAAGUGCUUCGAUUUUG 497 miR-373 GGGUGU miR-374 UUAUAAUACAACCUGAU 498miR-374 AAGUG

The present invention encompasses methods of diagnosing orprognosticating whether a subject has, or is at risk for developing, acancer and/or myeloproliferative disorder. The methods comprisedetermining the level of at least one miR gene product in a sample fromthe subject and comparing the level of the miR gene product in thesample to a control. As used herein, a “subject” can be any mammal thathas, or is suspected of having, a cancer and/or myeloproliferativedisorder. In a preferred embodiment, the subject is a human who has, oris suspected of having, a cancer, myeloproliferative disorder and/or aplatelet disorder.

The level of at least one miR gene product can be measured in cells of abiological sample obtained from the subject. For example, a tissuesample can be removed from a subject suspected of having cancer and/or amyeloproliferative disorder by conventional biopsy techniques. Inanother embodiment, a blood sample can be removed from the subject, andwhite blood cells can be isolated for DNA extraction by standardtechniques. In one embodiment, the blood or tissue sample is obtainedfrom the subject prior to initiation of radiotherapy, chemotherapy orother therapeutic treatment. A corresponding control tissue or bloodsample, or a control reference sample (e.g., obtained from a populationof control samples), can be obtained from unaffected tissues of thesubject, from a normal human individual or population of normalindividuals, or from cultured cells corresponding to the majority ofcells in the subject's sample. The control tissue or blood sample canthen processed along with the sample from the subject, so that thelevels of miR gene product produced from a given miR gene in cells fromthe subject's sample can be compared to the corresponding miR geneproduct levels from cells of the control sample. Alternatively, areference sample can be obtained and processed separately (e.g., at adifferent time) from the test sample and the level of a miR gene productproduced from a given miR gene in cells from the test sample can becompared to the corresponding miR gene product level from the referencesample.

In one embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “upregulated”). As used herein, expression of a miR gene product is“upregulated” when the amount of miR gene product in a cell or tissuesample from a subject is greater than the amount of the same geneproduct in a control (e.g., a reference standard, a control cell sample,a control tissue sample). In another embodiment, the level of the atleast one miR gene product in the test sample is less than the level ofthe corresponding miR gene product in the control sample (i.e.,expression of the miR gene product is “downregulated”). As used herein,expression of a miR gene is “downregulated” when the amount of miR geneproduct produced from that gene in a cell or tissue sample from asubject is less than the amount produced from the same gene in a controlcell or tissue sample. The relative miR gene expression in the controland normal samples can be determined with respect to one or more RNAexpression standards. The standards can comprise, for example, a zeromiR gene expression level, the miR gene expression level in a standardcell line, the miR gene expression level in unaffected tissues of thesubject, or the average level of miR gene expression previously obtainedfor a population of normal human controls (e.g., a control referencestandard).

An alteration (i.e., an increase or decrease) in the level of a miR geneproduct in the sample obtained from the subject, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof the presence of cancer and/or a myeloproliferative disorder in thesubject. In one embodiment, the level of the at least one miR geneproduct in the test sample is greater than the level of thecorresponding miR gene product in the control sample. miR gene productshaving higher expression levels in cancer cell lines (e.g., AMKL celllines) than control cells (e.g., in vitro CD34⁺-differentiatedmegakaryocytes) are described and exemplified herein (see, e.g., Example5). In one embodiment, the at least one miR gene product is selectedfrom the group consisting of miR-101, miR-126, miR-99a, miR-99-prec,miR-106, miR-339, miR-99b, miR-149, miR-33, miR-135, miR-20 andcombinations thereof. In another embodiment, the at least one miR geneproduct is selected from the group consisting of miR-101, miR-126,miR-106, miR-20 and miR-135 and combinations thereof. In yet anotherembodiment, the at least one miR gene product is selected from the groupconsisting of miR-106, miR-20 and miR-135 and combinations thereof. Asdescribed and exemplified herein, the increased expression of such miRgene products discriminates cancerous cells from correspondingnon-cancerous cells.

As described herein, the diagnostic and prognostic methods of theinvention can be used to diagnose or prognosticate cancers and/ormyeloproliferative disorders. In particular embodiments, the diagnosticand prognostic methods are used to diagnose or prognosticate a cancer ina subject, tissue sample, cell sample or fluid sample. The diagnosticand prognostic methods can be used to diagnose or prognosticate any typeof cancer. In particular embodiments, the diagnostic and prognosticmethods can be used to diagnose or prognosticate a leukemia. In oneembodiment, the leukemia that is diagnosed or prognosticated is acutemyeloid leukemia (e.g., acute megakaryoblastic leukemia). In otherembodiments, the diagnostic and prognostic methods can be used todiagnose or prognosticate multiple myeloma.

The diagnostic and prognostic methods of the invention can also be usedto diagnose or prognosticate hematologic malignancies (e.g.,myeloproliferative disorders). In one embodiment, the myeloproliferativedisorder that is diagnosed or prognosticated is selected from the groupconsisting of essential thrombocytemia (ET), polycythemia vera (PV),myelodisplasia, myelofibrosis (e.g., agnogenic myeloid metaplasia (AMM)(also referred to as idiopathic myelofibrosis)) and chronic myelogenousleukemia (CML).

In particular embodiments, the diagnostic, prognostic and therapeuticmethods of the invention can also be used to diagnose, prognosticateand/or treat platelet disorders (e.g., inherited platelet disorders).For example, the diagnostic, prognostic and therapeutic methods can beused to diagnose, prognosticate and/or treat defects in platelet-vesselwall interactions (i.e., disorders of adhesion). Such adhesion disordersinclude, e.g., von Willebrand disease (deficiency or defect in plasmavWF) and Bernard-Soulier syndrome (deficiency or defect in GPIb). Inother embodiments, the diagnostic, prognostic and therapeutic methodscan be used to diagnose, prognosticate and/or treat defects inplatelet-platelet interaction (i.e., disorders of aggregation). Suchaggregation disorders include, e.g., congenital afibrinogenemia(deficiency of plasma fibrinogen) and glanzmann thrombasthenia(deficiency or defect in GPIIb-IIIa). In other embodiments, thediagnostic, prognostic and therapeutic methods can be used to diagnose,prognosticate and/or treat disorders of platelet secretion andabnormalities of granules. Such disorders of platelet secretion andabnormalities of granules include, e.g., storage pool deficiency andQuebec platelet disorder. In yet other embodiments, the diagnostic,prognostic and therapeutic methods can be used to diagnose,prognosticate and/or treat disorders of platelet secretion and signaltransduction (primary secretion defects). Such primary secretion defectsinclude, e.g., defects in platelet-agonist interaction (receptordefects) (e.g., thromboxane A₂, collagen, ADP, epinephrine), defects inG-protein activation (e.g., Gαq deficiency, Gαs abnormalities, Gαideficiency), defects in phosphatidylinositol metabolism (e.g.,phospholipase C-2 deficiency), defects in calcium mobilization, defectsin protein phosphorylation (pleckstrin) PKC-y deficiency, andabnormalities in arachidonic acid pathways and thromboxane synthesis(e.g., cyclooxygenase deficiency, thromboxane synthase deficiency). Inother embodiments, the diagnostic, prognostic and therapeutic methodscan be used to diagnose, prognosticate and/or treat defects incytoskeletal regulation (e.g., Wiskott-Aldrich syndrome). In still otherembodiments, the diagnostic, prognostic and therapeutic methods can beused to diagnose, prognosticate and/or treat disorders of plateletcoagulant-protein interaction (membrane phospholipid defects) (e.g.,Scott syndrome). Other platelet disorders (e.g., inherited plateletdisorders) can also be diagnosed, prognosticated and/or treated usingthe methods of the invention.

The invention also provides methods of determining the prognosis of asubject with cancer and/or a myeloproliferative disorder. In thismethod, the level of at least one miR gene product, which is associatedwith a particular prognosis in cancer and/or a myeloproliferativedisorder (e.g., a good or positive prognosis, a poor or adverseprognosis), is measured in a test sample from the subject. An alteration(e.g., an increase, a decrease) in the level of the miR gene product inthe test sample, relative to the level of a corresponding miR geneproduct in a control sample, is indicative of the subject having acancer and/or myeloproliferative disorder with a particular prognosis.In one embodiment, the miR gene product is associated with an adverse(i.e., poor) prognosis. Examples of an adverse prognosis include, butare not limited to, low survival rate and rapid disease progression. Inone embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in a control sample (i.e., it is upregulated). In a particularembodiment, the at least one miR gene product that is upregulated isselected from the group consisting of miR-101, miR-126, miR-99a,miR-99-prec, miR-106, miR-339, miR-99b, miR-149, miR-33, miR-135, miR-20and combinations thereof. In another embodiment, the at least one miRgene product that is upregulated is selected from the group consistingof miR-101, miR-126, miR-106, miR-20 and miR-135 and combinationsthereof. In yet another embodiment, the at least one miR gene productthat is upregulated is selected from the group consisting of miR-106,miR-20 and miR-135 and combinations thereof. The increased expression ofsuch miR gene products can correlate with an adverse prognosis and theseverity of a subject's cancer and/or myeloproliferative disorder.

In certain embodiments of the diagnostic and prognostic methodsdescribed herein, the level of the at least one miR gene product ismeasured by reverse transcribing RNA from a test sample obtained fromthe subject to provide a set of target oligodeoxynucleotides,hybridizing the target oligodeoxynucleotides to a microarray thatcomprises miRNA-specific probe oligonucleotides to provide ahybridization profile for the test sample, and comparing the test samplehybridization profile to a hybridization profile generated from acontrol sample.

Identification of targets of particular miR gene products (e.g., thosemiR gene products exhibiting upregulated or downregulated expressionrelative to a control sample) can aid in elucidating mechanisms ofaction of microRNAs. As described and exemplified herein, particulartargets and putative targets of select microRNAs were identified (see,e.g., Tables 2, 3 and 5 and Exemplification). For example, thetranscription factor MAFB was identified as a target of mi-130a (Example2). Similarly, HOXA1 was identified as a target of miR-10a (Example 5).For both miRs, direct interaction of the miR with the 3′ UTR of itsrespective target was demonstrated (Examples 2 and 5). Moreover, aninverse relation in the expression of the miR and its respective targetwere demonstrated. Thus, expression of pre-miR-130a resulted indecreased expression of MAFB (see, e.g., FIG. 2C) while expression ofpre-miR-10a resulted in decreased expression of HOXA1 (see, e.g., FIGS.3C, 3F and 3G). Thus, in one embodiment, expression of target genes ofparticular microRNAs (e.g., those listed in Tables 2, 3 and 5) can beused to diagnose cancer and/or a myeloproliferative disorder. Suchtarget genes display inverse expression to the respective miR thattargets it. One of skill in the art can measure the expression levels ofany of these target genes using known methods and/or methods describedherein for measuring the expression levels of microRNAs (e.g.,quantitative or semi-quantitative RT-PCR, Northern blot analysis,solution hybridization detection, microarray analysis), without undueexperimentation. In particular embodiments, the target gene that ismeasured is MAFB or HOXA 1.

The level of the at least one miR gene product can be measured using avariety of techniques that are well known to those of skill in the art(e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis,solution hybridization detection). In a particular embodiment, the levelof at least one miR gene product is measured by reverse transcribing RNAfrom a test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides toone or more miRNA-specific probe oligonucleotides (e.g., a microarraythat comprises miRNA-specific probe oligonucleotides) to provide ahybridization profile for the test sample, and comparing the test samplehybridization profile to a hybridization profile generated from acontrol sample. An alteration in the signal of at least one miRNA in thetest sample relative to the control sample is indicative of the subjecteither having, or being at risk for developing cancer and/or amyeloproliferative disorder. In one embodiment, the signal of at leastone miRNA is upregulated, relative to the signal generated from thecontrol sample. In another embodiment, the signal of at least one miRNAis downregulated, relative to the signal generated from the controlsample. In a particular embodiment, the microarray comprisesmiRNA-specific probe oligonucleotides for a substantial portion of allknown human miRNAs (e.g., the miRNAs listed in Tables 1a and 1b plusother known or discovered miRNAs). In a further embodiment, themicroarray comprises miRNA-specific probe oligonucleotides for one ormore miRNAs selected from the group consisting of miR-101, miR-126,miR-99a, miR-99-prec, miR-106, miR-339, miR-99b, miR-149, miR-33,miR-135, miR-20 and a combination thereof. In one embodiment, themicroarray comprises miRNA-specific probe oligonucleotides for one ormore miRNAs selected from the group consisting of miR-101, miR-126,miR-106, miR-20, miR-135 and a combination thereof.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs and other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6× SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75× TNT at37° C. for 40 minutes. At positions on the array where the immobilizedprobe DNA recognizes a complementary target cDNA in the sample,hybridization occurs. The labeled target cDNA marks the exact positionon the array where binding occurs, allowing automatic detection andquantification. The output consists of a list of hybridization events,indicating the relative abundance of specific cDNA sequences, andtherefore the relative abundance of the corresponding complementarymiRs, in the patient sample. According to one embodiment, the labeledcDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeledprimer. The microarray is then processed by direct detection of thebiotin-containing transcripts using, e.g., Streptavidin-Alexa647conjugate, and scanned utilizing conventional scanning methods. Imageintensities of each spot on the array are proportional to the abundanceof the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool would allow for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a cancer and/or amyeloproliferative disorder is quantitatively reverse transcribed toprovide a set of labeled target oligodeoxynucleotides complementary tothe RNA in the sample. The target oligodeoxynucleotides are thenhybridized to a microarray comprising miRNA-specific probeoligonucleotides to provide a hybridization profile for the sample. Theresult is a hybridization profile for the sample representing theexpression pattern of miRNA in the sample. The hybridization profilecomprises the signal from the binding of the targetoligodeoxynucleotides from the sample to the miRNA-specific probeoligonucleotides in the microarray. The profile may be recorded as thepresence or absence of binding (signal vs. zero signal). Morepreferably, the profile recorded includes the intensity of the signalfrom each hybridization. The profile is compared to the hybridizationprofile generated from a normal (e.g., noncancerous,non-myeloproliferative disorder) control sample or reference sample. Analteration in the signal is indicative of the presence of, or propensityto develop, cancer in the subject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

The invention also provides methods of diagnosing whether a subject has,or is at risk for developing, a cancer and/or myeloproliferativedisorder with an adverse prognosis. In this method, the level of atleast one miR gene product, which is associated with an adverseprognosis in a cancer and/or myeloproliferative disorder, is measured byreverse transcribing RNA from a test sample obtained from the subject toprovide a set of target oligodeoxynucleotides. The targetoligodeoxynucleotides are then hybridized to one or more miRNA-specificprobe oligonucleotides (e.g., a microarray that comprises miRNA-specificprobe oligonucleotides) to provide a hybridization profile for the testsample, and the test sample hybridization profile is compared to ahybridization profile generated from a control sample. An alteration inthe signal of at least one miRNA in the test sample relative to thecontrol sample is indicative of the subject either having, or being atrisk for developing, a cancer and/or myeloproliferative disorder with anadverse prognosis. miRs suitable for use in this method include, e.g.,those that are upregulated in cancerous cells (e.g., AMKL cells).

In particular embodiments of the diagnostic, prognostic and therapeuticmethods of the invention, as well as the pharmaceutical compositions ofthe invention, the miR gene product is not one or more of let7a-2,let-7c, let-7g, let-71, miR-7-2, miR-7-3, miR-9, miR-9-1, miR-10a,miR-15a, miR-15b, miR-16-1, miR-16-2, miR-17-5p, miR-20a, miR-21,miR-24-1, miR-24-2, miR-25, miR-29b-2, miR-30, miR-30a-5p, miR-30c,miR-30d, miR-31, miR-32, miR-34, miR-34a, miR-34a prec, miR-34a-1,miR-34a-2, miR-92-2, miR-96, miR-99a, miR-99b prec, miR-100, miR-103,miR-106a, miR-107, miR-123, miR-124a-1, miR-125b-1, miR-125b-2,miR-126*, miR-127, miR-128b, miR-129, miR-129-1/2 prec, miR-132,miR-135-1, miR-136, miR-137, miR-141, miR-142-as, miR-143, miR-146,miR-148, miR-149, miR-153, miR-155, miR 159-1, miR-181, miR-181b-1,miR-182, miR-186, miR-191, miR-192, miR-195, miR-196-1, miR-196-1 prec,miR-196-2, miR-199a-1, miR-199a-2, miR-199b, miR-200b, miR-202, miR-203,miR-204, miR-205, miR-210, miR-211, miR-212, miR-214, miR-215, miR-217,miR-221 and/or miR-223.

As described herein, the level of a miR gene product in a sample can bemeasured using any technique that is suitable for detecting RNAexpression levels in a biological sample. Suitable techniques (e.g.,Northern blot analysis, RT-PCR, in situ hybridization) for determiningRNA expression levels in a biological sample (e.g., cells, tissues) arewell known to those of skill in the art. In a particular embodiment, thelevel of at least one miR gene product is detected using Northern blotanalysis. For example, total cellular RNA can be purified from cells byhomogenization in the presence of nucleic acid extraction buffer,followed by centrifugation. Nucleic acids are precipitated, and DNA isremoved by treatment with DNase and precipitation. The RNA molecules arethen separated by gel electrophoresis on agarose gels according tostandard techniques, and transferred to nitrocellulose filters. The RNAis then immobilized on the filters by heating. Detection andquantification of specific RNA is accomplished using appropriatelylabeled DNA or RNA probes complementary to the RNA in question. See, forexample, Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7,the entire disclosure of which is incorporated by reference.

Suitable probes (e.g., DNA probes, RNA probes) for Northern blothybridization of a given miR gene product can be produced from thenucleic acid sequences provided in Table 1a and Table 1b and include,but are not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% complementarity to a miR gene product of interest,as well as probes that have complete complementarity to a miR geneproduct of interest. Methods for preparation of labeled DNA and RNAprobes, and the conditions for hybridization thereof to targetnucleotide sequences, are described in Molecular Cloning: A LaboratoryManual, J. Sambrook et al., eds., 2nd edition, Cold Spring HarborLaboratory Press, 1989, Chapters 10 and 11, the disclosures of which areincorporated herein by reference.

For example, the nucleic acid probe can be labeled with, e.g., aradionuclide, such as ³H, ³²P, ³³P, ¹⁴C, ³⁵S; a heavy metal; a ligandcapable of functioning as a specific binding pair member for a labeledligand (e.g., biotin, avidin or an antibody); a fluorescent molecule; achemiluminescent molecule; an enzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing³²P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare ³²P-labeled nucleic acid probes with aspecific activity well in excess of 10⁸ cpm/microgram. Autoradiographicdetection of hybridization can then be performed by exposing hybridizedfilters to photographic film. Densitometric scanning of the photographicfilms exposed by the hybridized filters provides an accurate measurementof miR gene transcript levels. Using another approach, miR genetranscript levels can be quantified by computerized imaging systems,such as the Molecular Dynamics 400-B 2D Phosphorimager available fromAmersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin and antibodies (e.g., anti-biotinantibodies) coupled to fluorescent dyes or enzymes that produce colorreactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique and involves depositing whole cellsonto a microscope cover slip and probing the nucleic acid content of thecell with a solution containing radioactive or otherwise labeled nucleicacid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference. Suitable probes for in situhybridization of a given miR gene product can be produced from thenucleic acid sequences provided in Table 1a and Table 1b, and include,but are not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% complementarity to a miR gene product of interest,as well as probes that have complete complementarity to a miR geneproduct of interest, as described above.

The relative number of miR gene transcripts in cells can also bedetermined by reverse transcription of miR gene transcripts, followed byamplification of the reverse-transcribed transcripts by polymerase chainreaction (RT-PCR), for example, as exemplified herein. The levels of miRgene transcripts can be quantified in comparison with an internalstandard, for example, the level of mRNA from a “housekeeping” genepresent in the same sample. A suitable “housekeeping” gene for use as aninternal standard includes, e.g., U6 small nuclear RNA, myosin orglyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods for performingquantitative and semi-quantitative RT-PCR, and variations thereof, arewell known to those of skill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a cancer and/or myeloproliferative disorder. Assessingcancer-specific expression levels for hundreds of miR genes or geneproducts is time consuming and requires a large amount of total RNA(e.g., at least 20 μg for each Northern blot) and autoradiographictechniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotide) probes that are specificfor a set of miR genes. Using such a microarray, the expression level ofmultiple microRNAs in a biological sample can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression level incancer cells and/or cells exhibiting a myeloproliferative disorder. Asused herein, “probe oligonucleotide” or “probe oligodeoxynucleotide”refers to an oligonucleotide that is capable of hybridizing to a targetoligonucleotide. “Target oligonucleotide” or “targetoligodeoxynucleotide” refers to a molecule to be detected (e.g., viahybridization). By “miR-specific probe oligonucleotide” or “probeoligonucleotide specific for a miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal tissue, cell or fluid samples may be distinguished fromcorresponding cancerous and/or myeloproliferative disorder-exhibitingtissue, cell or fluid samples. Within cancerous and/ormyeloproliferative disorder-exhibiting tissue, cell or fluid samples,different prognosis states (for example, good or poor long term survivalprospects) may be determined. By comparing expression profiles ofcancerous and/or myeloproliferative disorder-exhibiting tissue, cell orfluid samples in different states, information regarding which genes areimportant (including both upregulation and downregulation of genes) ineach of these states is obtained. The identification of sequences thatare differentially expressed in cancerous and/or myeloproliferativedisorder-exhibiting tissue, cell or fluid samples, as well asdifferential expression resulting in different prognostic outcomes,allows the use of this information in a number of ways. For example, aparticular treatment regime may be evaluated (e.g., to determine whethera chemotherapeutic drug acts to improve the long-term prognosis in aparticular subject). Similarly, diagnosis may be done or confirmed bycomparing samples from a subject with known expression profiles.Furthermore, these gene expression profiles (or individual genes) allowscreening of drug candidates that suppress the cancer and/ormyeloproliferative disorder expression profile or convert a poorprognosis profile to a better prognosis profile.

Without wishing to be bound by any one theory, it is believed thatalterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to aberrant megakaryocytic differentiation and/orthe formation of cancer, a myeloproliferative disorder and/or a plateletdisorder. Therefore, altering the level of the miR gene product (e.g.,by decreasing the level of a miR that is upregulated in cancerous and/ormyeloproliferative disorder-exhibiting cells, by increasing the level ofa miR that is downregulated in cancerous and/or myeloproliferativedisorder-exhibiting cells) may successfully treat the cancer,myeloproliferative disorder and/or platelet disorder.

Accordingly, the present invention encompasses methods of treating acancer and/or myeloproliferative disorder in a subject, wherein at leastone miR gene product is deregulated (e.g., downregulated, upregulated)in the cells (e.g., cancerous cells and/or myeloproliferativedisorder-exhibiting cells) of the subject. In one embodiment, the levelof at least one miR gene product in a test sample (e.g., a samplecomprising cancerous and/or myeloproliferative disorder-exhibitingtissues, cells or fluid) is greater than the level of the correspondingmiR gene product in a control or reference sample. In anotherembodiment, the level of at least one miR gene product in a test sample(e.g., a sample comprising cancerous and/or myeloproliferativedisorder-exhibiting tissues, cells or fluid) is less than the level ofthe corresponding miR gene product in a control sample. When the atleast one isolated miR gene product is downregulated in the test sample(e.g., a sample comprising cancerous and/or myeloproliferativedisorder-exhibiting tissues, cells or fluid), the method comprisesadministering an effective amount of the at least one isolated miR geneproduct, or an isolated variant or biologically-active fragment thereof,such that proliferation of the cancerous and/or myeloproliferativedisorder-exhibiting cells in the subject is inhibited. For example, whena miR gene product is downregulated in a cancer cell in a subject,administering an effective amount of an isolated miR gene product to thesubject can inhibit proliferation of the cancer cell. The isolated miRgene product that is administered to the subject can be identical to anendogenous wild-type miR gene product (e.g., a miR gene product shown inTable 1a or Table 1b) that is downregulated in the cancer cell or it canbe a variant or biologically-active fragment thereof. As defined herein,a “variant” of a miR gene product refers to a miRNA that has less than100% identity to a corresponding wild-type miR gene product andpossesses one or more biological activities of the correspondingwild-type miR gene product. Examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule (e.g., inhibiting translation of a target RNA molecule,modulating the stability of a target RNA molecule, inhibiting processingof a target RNA molecule) and inhibition of a cellular processassociated with cancer and/or a myeloproliferative disorder (e.g., celldifferentiation, cell growth, cell death). These variants includespecies variants and variants that are the consequence of one or moremutations (e.g., a substitution, a deletion, an insertion) in a miRgene. In certain embodiments, the variant is at least about 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% identical to a corresponding wild-typemiR gene product.

As defined herein, a “biologically-active fragment” of a miR geneproduct refers to an RNA fragment of a miR gene product that possessesone or more biological activities of a corresponding wild-type miR geneproduct. As described above, examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule and inhibition of a cellular process associated with cancerand/or a myeloproliferative disorder. In certain embodiments, thebiologically-active fragment is at least about 5, 7, 10, 12, 15, or 17nucleotides in length. In a particular embodiment, an isolated miR geneproduct can be administered to a subject in combination with one or moreadditional anti-cancer treatments. Suitable anti-cancer treatmentsinclude, but are not limited to, chemotherapy, radiation therapy andcombinations thereof (e.g., chemoradiation).

When the at least one isolated miR gene product is upregulated in thecancer cells, the method comprises administering to the subject aneffective amount of a compound that inhibits expression of the at leastone miR gene product, such that proliferation of the cancer and/ormyeloproliferative disorder-exhibiting cells is inhibited. Suchcompounds are referred to herein as miR gene expression-inhibitioncompounds. Examples of suitable miR gene expression-inhibition compoundsinclude, but are not limited to, those described herein (e.g.,double-stranded RNA, antisense nucleic acids and enzymatic RNAmolecules). In a particular embodiment, a miR gene expression-inhibitingcompound can be administered to a subject in combination with one ormore additional anti-cancer treatments. Suitable anti-cancer treatmentsinclude, but are not limited to, chemotherapy, radiation therapy andcombinations thereof (e.g., chemoradiation).

As described, when the at least one isolated miR gene product isupregulated in cancer cells (e.g., AMKL cells), the method comprisesadministering to the subject an effective amount of at least onecompound for inhibiting expression of the at least one miR gene product,such that proliferation of cancer cells is inhibited. In one embodiment,the compound for inhibiting expression of the at least one miR geneproduct inhibits a miR gene product selected from the group consistingof miR-101, miR-126, miR-99a, miR-99-prec, miR-106, miR-339, miR-99b,miR-149, miR-33, miR-135, miR-20 and a combination thereof. In anotherembodiment, the compound for inhibiting expression of the at least onemiR gene product inhibits a miR gene product selected from the groupconsisting of miR-101, miR-126, miR-106, miR-20, miR-135 and acombination thereof. In yet another embodiment, the compound forinhibiting expression of the at least one miR gene product inhibits amiR gene product selected from the group consisting of miR-106, miR-20,miR-135 and a combination thereof.

As described and exemplified herein, the transcription factor MAFB,which is upregulated in megakaryocytic differentiation, is a target ofmiR-130a. Moreover, an inverse relation in the expression of miR-130aand its respective target were demonstrated. Thus, expression ofpre-miR-130a resulted in decreased expression of MAFB (see, e.g., FIG.2C). MAFB is known to be deregulated in cancer (e.g., multiple myelomaand acute myeloid leukemia). For example, ectopic expression of MAFB hasbeen observed in human myeloma cells carrying (14;20)(q32;q11)chromosomal translocations (Hanamura, I., et al. (2001) Jpn. J. CancerRes. 92(6):638-644 (2001)). Accordingly, in one embodiment, theinvention is a method of treating a cancer and/or myeloproliferativedisorder in a subject comprising administering an effective amount of atleast one miR gene product or an isolated variant or biologically-activefragment thereof to the subject, wherein:

the cancer and/or myeloproliferative disorder is associated withoverexpression of a MAFB gene product; and

the at least one miR gene product binds to, and decreases expression of,the MAFB gene product.

In one embodiment, the at least one miR gene product or isolated variantor biologically-active fragment thereof comprises a nucleotide sequencethat is complementary to a nucleotide sequence in the MAFB gene product(e.g., complementary to the 3′ UTR of MAFB). In a particular embodiment,the at least one miR gene product is miR-130a or an isolated variant orbiologically-active fragment thereof.

Also as described and exemplified herein, mRNA of HOXA1, one of themembers of the HOX family of proteins, is upregulated 7-fold inmegakaryocytic differentiation (see, e.g., Example 4). Moreover, HOXA1is a target of miR-10a and its expression is inversely related to theexpression of miR-10a. Thus, expression of pre-miR-10a resulted indecreased expression of HOXA1 (see, e.g., FIGS. 3C, 3F and 3G). HOXA1.Expression of HOXA1 has been demonstrated to be sufficient to result inthe oncogenic transformation of immortalized human mammary epithelialcells with aggressive in vivo tumor formation (Zhang, X., et al., (2002)J. Biol. Chem. 278(9):7580-7590). Further, forced expression of HOXA1 inmammary carcinoma cells, in a Bcl-2-dependent manner, resulted in adramatic enhancement of anchorage-independent proliferation and colonyformation in soft agar. Id. Accordingly, in one embodiment, theinvention is a method of treating a cancer and/or myeloproliferativedisorder in a subject comprising administering an effective amount of atleast one miR gene product or an isolated variant or biologically-activefragment thereof to the subject, wherein:

the cancer and/or myeloproliferative disorder is associated withoverexpression of a HOXA1 gene product; and

the at least one miR gene product binds to, and decreases expression of,the HOXA1 gene product.

In one embodiment, the at least one miR gene product or isolated variantor biologically-active fragment thereof comprises a nucleotide sequencethat is complementary to a nucleotide sequence in the HOXA1 gene product(e.g., complementary to the 3′ UTR of HOXA1). In a particularembodiment, the at least one miR gene product is miR-10a or an isolatedvariant or biologically-active fragment thereof.

In a related embodiment, the methods of treating cancer and/or amyeloproliferative disorder in a subject additionally comprise the stepof first determining the amount of at least one miR gene product in asample from the subject, and comparing that level of the miR geneproduct to the level of a corresponding miR gene product in a control.If expression of the miR gene product is deregulated (e.g.,downregulated, upregulated) in the sample from the subject, the methodsfurther comprise altering the amount of the at least one miR geneproduct expressed in the sample from the subject. In one embodiment, theamount of the miR gene product expressed in the sample from the subjectis less than the amount of the miR gene product expressed in thecontrol, and an effective amount of the miR gene product, or an isolatedvariant or biologically-active fragment thereof, is administered to thesubject. In another embodiment, the amount of the miR gene productexpressed in the sample from the subject is greater than the amount ofthe miR gene product expressed in the control, and an effective amountof at least one compound for inhibiting expression of the at least onemiR gene is administered to the subject. Suitable miRs and compoundsthat inhibit expression of miR genes include, for example, thosedescribed herein.

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, cancer and/or a myeloproliferative disorder, includingpreventing or delaying the onset of the disease symptoms, and/orlessening the severity or frequency of symptoms of the disease orcondition. The terms “subject”, “patient” and “individual” are definedherein to include animals, such as mammals, including, but not limitedto, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guineapigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent,or murine species. In a preferred embodiment, the animal is a human.

As used herein, an “effective amount” of an isolated miR gene product isan amount sufficient to inhibit proliferation of cells (e.g., cancerouscells, cells exhibiting a myeloproliferative disorder) in a subjectsuffering from cancer and/or a myeloproliferative disorder. One skilledin the art can readily determine an effective amount of a miR geneproduct to be administered to a given subject, by taking into accountfactors, such as the size and weight of the subject; the extent ofdisease penetration; the age, health and sex of the subject; the routeof administration; and whether the administration is regional orsystemic.

For example, an effective amount of an isolated miR gene product can bebased on the approximate weight of a tumor mass to be treated. Theapproximate weight of a tumor mass can be determined by calculating theapproximate volume of the mass, wherein one cubic centimeter of volumeis roughly equivalent to one gram. An effective amount of the isolatedmiR gene product based on the weight of a tumor mass can be in the rangeof about 10-500 micrograms/gram of tumor mass. In certain embodiments,the tumor mass can be at least about 10 micrograms/gram of tumor mass,at least about 60 micrograms/gram of tumor mass or at least about 100micrograms/gram of tumor mass.

An effective amount of an isolated miR gene product can also be based onthe approximate or estimated body weight of a subject to be treated.Preferably, such effective amounts are administered parenterally orenterally, as described herein. For example, an effective amount of theisolated miR gene product that is administered to a subject can rangefrom about 5-3000 micrograms/kg of body weight, from about 700-1000micrograms/kg of body weight, or greater than about 1000 micrograms/kgof body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for the administration of an isolated miR gene product to agiven subject. For example, a miR gene product can be administered tothe subject once (e.g., as a single injection or deposition).Alternatively, a miR gene product can be administered once or twicedaily to a subject for a period of from about three to abouttwenty-eight days, more particularly from about seven to about ten days.In a particular dosage regimen, a miR gene product is administered oncea day for seven days. Where a dosage regimen comprises multipleadministrations, it is understood that the effective amount of the miRgene product administered to the subject can comprise the total amountof gene product administered over the entire dosage regimen.

As used herein, an “isolated” miR gene product is one that issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or a miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in a substantially-purified form, or can exist ina cell into which the miR gene product has been delivered. Thus, a miRgene product that is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. A miR gene productproduced inside a cell from a miR precursor molecule is also consideredto be an “isolated” molecule. According to the invention, the isolatedmiR gene products described herein can be used for the manufacture of amedicament for treating cancer and/or a myeloproliferative disorder in asubject (e.g., a human).

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cells(e.g., cancerous cells, cells exhibiting a myeloproliferative disorder).

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products that are expressed from recombinantplasmids can also be delivered to, and expressed directly in, cells(e.g., cancerous cells, cells exhibiting a myeloproliferative disorder).The use of recombinant plasmids to deliver the miR gene products tocells (e.g., cancerous cells, cells exhibiting a myeloproliferativedisorder) is discussed in more detail below.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell. Other suitable processing systems include, e.g., the invitro Drosophila cell lysate system (e.g., as described in U.S.Published Patent Application No. 2002/0086356 to Tuschl et al., theentire disclosure of which is incorporated herein by reference) and theE. coli RNAse III system (e.g., as described in U.S. Published PatentApplication No. 2004/0014113 to Yang et al., the entire disclosure ofwhich is incorporated herein by reference).

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference.

In one embodiment, a plasmid expressing the miR gene products comprisesa sequence encoding a miR precursor RNA under the control of the CMVintermediate-early promoter. As used herein, “under the control” of apromoter means that the nucleic acid sequences encoding the miR geneproduct are located 3′ of the promoter, so that the promoter caninitiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cells (e.g., cancerouscells, cells exhibiting a myeloproliferative disorder). The use ofrecombinant viral vectors to deliver the miR gene products to cells(e.g., cancerous cells, cells exhibiting a myeloproliferative disorder)is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR gene products and any suitable promoter for expressingthe RNA sequences. Suitable promoters include, but are not limited to,the U6 or H1 RNA pol III promoter sequences, or the cytomegaloviruspromoters. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the miRgene products in a cancer cell.

Any viral vector capable of accepting the coding sequences for the miRgene products can be used; for example, vectors derived from adenovirus(AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.The tropism of the viral vectors can be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses, or by substituting different viral capsid proteins, asappropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorsthat express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz, J. E., et al. (2002), J. Virol.76:791-801, the entire disclosure of which is incorporated herein byreference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingRNA into the vector, methods of delivering the viral vector to the cellsof interest, and recovery of the expressed RNA products are within theskill in the art. See, for example, Dornburg (1995), Gene Therapy2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are incorporated herein by reference.

Particularly suitable viral vectors are those derived from AV and AAV. Asuitable AV vector for expressing the miR gene products, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia et al. (2002), Nat.Biotech. 20:1006-1010, the entire disclosure of which is incorporatedherein by reference. Suitable AAV vectors for expressing the miR geneproducts, methods for constructing the recombinant AAV vector, andmethods for delivering the vectors into target cells are described inSamulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J.Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are incorporated herein byreference. In one embodiment, the miR gene products are expressed from asingle recombinant AAV vector comprising the CMV intermediate earlypromoter.

In a certain embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding a miR precursor RNA inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. As used herein, “in operable connection witha polyT termination sequence” means that the nucleic acid sequencesencoding the sense or antisense strands are immediately adjacent to thepolyT termination signal in the 5′ direction. During transcription ofthe miR sequences from the vector, the polyT termination signals act toterminate transcription.

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound that inhibits miR expressioncan be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the precursor and/or active,mature form of miR gene product after treatment is less than the amountproduced prior to treatment. One skilled in the art can readilydetermine whether miR expression has been inhibited in cells (e.g.,cancerous cells, cells exhibiting a myeloproliferative disorder), using,for example, the techniques for determining miR transcript leveldiscussed herein. Inhibition can occur at the level of gene expression(i.e., by inhibiting transcription of a miR gene encoding the miR geneproduct) or at the level of processing (e.g., by inhibiting processingof a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of cells(e.g., cancerous cells, cells exhibiting a myeloproliferative disorder)in a subject suffering from cancer and/or a myeloproliferative disorder.One skilled in the art can readily determine an effective amount of amiR expression-inhibiting compound to be administered to a givensubject, by taking into account factors, such as the size and weight ofthe subject; the extent of disease penetration; the age, health and sexof the subject; the route of administration; and whether theadministration is regional or systemic.

For example, an effective amount of the expression-inhibiting compoundcan be based on the approximate weight of a tumor mass to be treated, asdescribed herein. An effective amount of a compound that inhibits miRexpression can also be based on the approximate or estimated body weightof a subject to be treated, as described herein.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject, as described herein. Suitable compounds for inhibitingmiR gene expression include double-stranded RNA (such as short- orsmall-interfering RNA or “siRNA”), antisense nucleic acids, andenzymatic RNA molecules, such as ribozymes. Each of these compounds canbe targeted to a given miR gene product and interfere with theexpression (e.g., by inhibiting translation, by inducing cleavage and/ordegradation) of the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example, at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence thatis substantially identical to a nucleic acid sequence contained withinthe target miR gene product.

As used herein, a nucleic acid sequence in an siRNA that is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

The siRNA can also be altered RNA that differs from naturally-occurringRNA by the addition, deletion, substitution and/or alteration of one ormore nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA or to one ormore internal nucleotides of the siRNA, or modifications that make thesiRNA resistant to nuclease digestion, or the substitution of one ormore nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in certainembodiments, the siRNA comprises at least one 3′ overhang of from 1 toabout 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, from 1 to about 5 nucleotides inlength, from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. In a particular embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as described abovefor the isolated miR gene products. Exemplary methods for producing andtesting dsRNA or siRNA molecules are described in U.S. Published PatentApplication No. 2002/0173478 to Gewirtz and in U.S. Published PatentApplication No. 2004/0018176 to Reich et al., the entire disclosures ofboth of which are incorporated herein by reference.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA,RNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, peptide nucleic acids (PNA)) that generally comprise anucleic acid sequence complementary to a contiguous nucleic acidsequence in a miR gene product. The antisense nucleic acid can comprisea nucleic acid sequence that is 50-100% complementary, 75-100%complementary, or 95-100% complementary to a contiguous nucleic acidsequence in a miR gene product. Nucleic acid sequences of particularhuman miR gene products are provided in Table 1a and Table 1b. Withoutwishing to be bound by any theory, it is believed that the antisensenucleic acids activate RNase H or another cellular nuclease that digeststhe miR gene product/antisense nucleic acid duplex.

Antisense nucleic acids can also contain modifications to the nucleicacid backbone or to the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators, such as acridine, orone or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing are within the skill in the art; see, e.g.,Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 toWoolf et al., the entire disclosures of which are incorporated herein byreference.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in a miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid foruse in the present methods is a ribozyme.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing dsRNA or siRNA molecules are described inWerner and Uhlenbeck (1995), Nucleic Acids Res. 23:2092-96; Hammann etal. (1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat.No. 4,987,071 to Cech et al, the entire disclosures of which areincorporated herein by reference.

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cells (e.g., cancerous cells, cells exhibiting a myeloproliferativedisorder) in a subject who has a cancer and/or a myeloproliferativedisorder. As used herein, to “inhibit the proliferation of cancerouscells or cells exhibiting a myeloproliferative disorder” means to killthe cells, or permanently or temporarily arrest or slow the growth ofthe cells. Inhibition of cell proliferation can be inferred if thenumber of such cells in the subject remains constant or decreases afteradministration of the miR gene products or miR geneexpression-inhibiting compounds. An inhibition of proliferation ofcancerous cells or cells exhibiting a myeloproliferative disorder canalso be inferred if the absolute number of such cells increases, but therate of tumor growth decreases.

The number of cancer cells in the body of a subject can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The size of a tumor mass can be ascertained by direct visualobservation, or by diagnostic imaging methods, such as X-ray, magneticresonance imaging, ultrasound, and scintigraphy. Diagnostic imagingmethods used to ascertain size of the tumor mass can be employed with orwithout contrast agents, as is known in the art. The size of a tumormass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

The miR gene products or miR gene expression-inhibiting compounds can beadministered to a subject by any means suitable for delivering thesecompounds to cells (e.g., cancer cells, cells exhibiting amyeloproliferative disorder) of the subject. For example, the miR geneproducts or miR expression-inhibiting compounds can be administered bymethods suitable to transfect cells of the subject with these compounds,or with nucleic acids comprising sequences encoding these compounds. Inone embodiment, the cells are transfected with a plasmid or viral vectorcomprising sequences encoding at least one miR gene product or miR geneexpression-inhibiting compound.

Transfection methods for eukaryotic cells are well known in the art, andinclude, e.g., direct injection of the nucleic acid into the nucleus orpronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor-mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

For example, cells can be transfected with a liposomal transfercompound, e.g., DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results may be achieved with 0.1-100 micrograms of nucleicacid/10⁵ cells. For example, a ratio of about 0.5 micrograms of plasmidvector in 3 micrograms of DOTAP per 10⁵ cells can be used.

A miR gene product or miR gene expression-inhibiting compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and direct injection into the tumor.

In the present methods, a miR gene product or miR gene productexpression-inhibiting compound can be administered to the subject eitheras naked RNA, in combination with a delivery reagent, or as a nucleicacid (e.g., a recombinant plasmid or viral vector) comprising sequencesthat express the miR gene product or miR gene expression-inhibitingcompound. Suitable delivery reagents include, e.g., the Mirus TransitTKO lipophilic reagent; LIPOFECTIN; lipofectamine; cellfectin;polycations (e.g., polylysine) and liposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR gene products or miR gene expression-inhibiting compounds, andtechniques for delivering such plasmids and vectors to cancer cells, arediscussed herein and/or are well known in the art.

In a particular embodiment, liposomes are used to deliver a miR geneproduct or miR gene expression-inhibiting compound (or nucleic acidscomprising sequences encoding them) to a subject. Liposomes can alsoincrease the blood half-life of the gene products or nucleic acids.Suitable liposomes for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors, such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are incorporated herein byreference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands that bind toreceptors prevalent in cancer cells, such as monoclonal antibodies thatbind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In a particularly preferred embodiment, a liposomeof the invention can comprise both an opsonization-inhibition moiety anda ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization-inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is incorporated herein byreference.

Opsonization-inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) or derivatives thereof; e.g.,methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers, such aspolyacrylamide or poly N-vinyl pyrrolidone; linear, branched, ordendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g.,polyvinylalcohol and polyxylitol to which carboxylic or amino groups arechemically linked, as well as gangliosides, such as ganglioside GM1.Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof,are also suitable. In addition, the opsonization-inhibiting polymer canbe a block copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. Theopsonization-inhibiting polymers can also be natural polysaccharidescontaining amino acids or carboxylic acids, e.g., galacturonic acid,glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid,neuraminic acid, alginic acid, carrageenan; aminated polysaccharides oroligosaccharides (linear or branched); or carboxylated polysaccharidesor oligosaccharides, e.g., reacted with derivatives of carbonic acidswith resultant linking of carboxylic groups. Preferably, theopsonization-inhibiting moiety is a PEG, PPG, or a derivative thereof.Liposomes modified with PEG or PEG-derivatives are sometimes called“PEGylated liposomes.”

The opsonization-inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example, solid tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miR gene products or miR gene expression-inhibition compounds (ornucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression-inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating cancer and/or amyeloproliferative disorder.

In one embodiment, the pharmaceutical composition of the inventioncomprises at least one miR expression-inhibition compound and apharmaceutically-acceptable carrier. In a particular embodiment, the atleast one miR expression-inhibition compound is specific for a miR geneproduct whose expression is greater in cancer cells than control cells(i.e., it is upregulated). In another embodiment, the miRexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting of miR-101, miR-126,miR-99a, miR-99-prec, miR-106, miR-339, miR-99b, miR-149, miR-33,miR-135 and miR-20. In another embodiment, the miR expression-inhibitioncompound is specific for one or more miR gene products selected from thegroup consisting of miR-101, miR-126, miR-106, miR-20, and miR-135. Inyet another embodiment, the miR expression-inhibition compound isspecific for one or more miR gene products selected from the groupconsisting of miR-106, miR-20 and miR-135.

In other embodiments, the pharmaceutical compositions comprise aneffective amount of at least one miR gene product, or an isolatedvariant or biologically-active fragment thereof, and apharmaceutically-acceptable carrier. In one embodiment, the invention isa pharmaceutical composition for treating a cancer and/or amyeloproliferative disorder, wherein the cancer and/ormyeloproliferative disorder is associated with overexpression of a MAFBgene product. In this embodiment, the pharmaceutical compositioncomprises at least one miR gene product that binds to, and decreasesexpression of, the MAFB gene product. In a particular embodiment, the atleast one miR gene product comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in the MAFB gene product. Inanother embodiment, the at least one miR gene product is miR-130a or anisolated variant or biologically-active fragment thereof.

In one embodiment, the invention is a pharmaceutical composition fortreating a cancer and/or a myeloproliferative disorder, wherein thecancer and/or myeloproliferative disorder is associated withoverexpression of a HOXA1 gene product. In this embodiment, thepharmaceutical composition comprises at least one miR gene product thatbinds to, and decreases expression of, the HOXA1 gene product. In aparticular embodiment, the at least one miR gene product comprises anucleotide sequence that is complementary to a nucleotide sequence inthe HOXA1 gene product. In another embodiment, the at least one miR geneproduct is miR-10a or an isolated variant or biologically-activefragment thereof.

Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical compositions” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example, as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is incorporatedherein by reference.

The present pharmaceutical compositions comprise at least one miR geneproduct or miR gene expression-inhibition compound (or at least onenucleic acid comprising a sequence encoding the miR gene product or miRgene expression-inhibition compound) (e.g., 0.1 to 90% by weight), or aphysiologically-acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. In certain embodiments, thepharmaceutical composition of the invention additionally comprises oneor more anti-cancer agents (e.g., chemotherapeutic agents). Thepharmaceutical formulations of the invention can also comprise at leastone miR gene product or miR gene expression-inhibition compound (or atleast one nucleic acid comprising a sequence encoding the miR geneproduct or miR gene expression-inhibition compound), which areencapsulated by liposomes and a pharmaceutically-acceptable carrier. Inone embodiment, the pharmaceutical composition comprises a miR gene orgene product that is not miR-15, miR-16, miR-143 and/or miR-145.

Especially suitable pharmaceutically-acceptable carriers are water,buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronicacid and the like.

In a particular embodiment, the pharmaceutical compositions of theinvention comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisinga sequence encoding the miR gene product or miR geneexpression-inhibition compound) that is resistant to degradation bynucleases. One skilled in the art can readily synthesize nucleic acidsthat are nuclease resistant, for example by incorporating one or moreribonucleotides that is modified at the 2′-position into the miR geneproduct. Suitable 2′-modified ribonucleotides include those modified atthe 2′-position with fluoro, amino, alkyl, alkoxy and O-allyl.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression-inhibition compound (or at least one nucleic acidcomprising a sequence encoding the miR gene product or miR geneexpression-inhibition compound) encapsulated in a liposome as describedabove, and a propellant. A carrier can also be included as desired;e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further compriseone or more anti-cancer agents. In a particular embodiment, thecompositions comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisinga sequence encoding the miR gene product or miR geneexpression-inhibition compound) and at least one chemotherapeutic agent.Chemotherapeutic agents that are suitable for the methods of theinvention include, but are not limited to, DNA-alkylating agents,anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizingagents, tubulin destabilizing agents, hormone antagonist agents,topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors,CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinaseinhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acidsaptamers, and molecularly-modified viral, bacterial and exotoxic agents.Examples of suitable agents for the compositions of the presentinvention include, but are not limited to, cytidine arabinoside,methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin),cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin,methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine,camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide,oxaliplatin, irinotecan, topotecan, leucovorin, carmustine,streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab,daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine,docetaxel and FOLFOX4.

The invention also encompasses methods of identifying an anti-canceragent, comprising providing a test agent to a cell and measuring thelevel of at least one miR gene product in the cell. In one embodiment,the method comprises providing a test agent to a cell and measuring thelevel of at least one miR gene product associated with increasedexpression levels in cancer cells (e.g., in AMKL cells). A decrease inthe level of the miR gene product that is associated with increasedexpression levels in cancer, relative to a suitable control (e.g., thelevel of the miR gene product in control cells), is indicative of thetest agent being an anti-cancer agent. In a particular embodiment, theat least one miR gene product associated with increased expressionlevels in cancer cells is selected from the group consisting of miR-101,miR-126, miR-99a, miR-99-prec, miR-106, miR-339, miR-99b, miR-149,miR-33, miR-135 and miR-20. In another embodiment, the at least one miRgene product associated with increased expression levels in cancer cellsis selected from the group consisting of miR-101, miR-126, miR-106,miR-20 and miR-135. In yet another embodiment, the at least one miR geneproduct associated with increased expression levels in cancer cells isselected from the group consisting of miR-106, miR-20 and miR-135. Inone embodiment, the miR gene product is not one or more of let7a-2,let-7c, let-7g, let-71, miR-7-2, miR-7-3, miR-9, miR-9-1, miR-10a,miR-15a, miR-15b, miR-16-1, miR-16-2, miR-17-5p, miR-20a, miR-21,miR-24-1, miR-24-2, miR-25, miR-29b-2, miR-30, miR-30a-5p, miR-30c,miR-30d, miR-31, miR-32, miR-34, miR-34a, miR-34a prec, miR-34a-1,miR-34a-2, miR-92-2, miR-96, miR-99a, miR-99b prec, miR-100, miR-103,miR-106a, miR-107, miR-123, miR-124a-1, miR-125b-1, miR-125b-2,miR-126*, miR-127, miR-128b, miR-129, miR-129-1/2 prec, miR-132,miR-135-1, miR-136, miR-137, miR-141, miR-142-as, miR-143, miR-146,miR-148, miR-149, miR-153, miR-155, miR 159-1, miR-181, miR-181b-1,miR-182, miR-186, miR-191, miR-192, miR-195, miR-196-1, miR-196-1 prec,miR-196-2, miR-199a-1, miR-199a-2, miR-199b, miR-200b, miR-202, miR-203,miR-204, miR-205, miR-210, miR-211, miR-212, miR-214, miR-215, miR-217,miR-221 and/or miR-223.

In one embodiment, the method comprises providing a test agent to a celland measuring the level of at least one miR gene product associated withdecreased expression levels in cancerous cells. An increase in the levelof the miR gene product in the cell, relative to a suitable control(e.g., the level of the miR gene product in a control cell), isindicative of the test agent being an anti-cancer agent.

Suitable agents include, but are not limited to drugs (e.g., smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art. Several of thesemethods are also described herein.

The invention will now be illustrated by the following non-limitingexamples.

EXEMPLIFICATION

Unless otherwise noted, the following materials and methods were used inthe Examples.

Material and Methods

Cell Lines and Human CD34⁺Cells.

The human chronic myeloid leukemia (CML) blast crisis cell lines K-562and MEG-01 were obtained from American Type Tissue Culture (ATCC,Manassas, Va.) and maintained in RPMI 1640 (GIBCO, Carlsbad, Calif.)containing 10% FBS with penicillin-gentamycin at 37° C. with 5% CO2. Thehuman megakaryoblastic leukemia cells UT-7, and CMK, and the chronicmyeloid leukemia (CML) in blast crisis LAMA were obtained from DSMZ(Braunsweig, Germany). All cells were maintained in RPMI medium 1640with 20% FBS and antibiotics, except UT-7 which is factor-dependent andwas cultured in MEM-□ with 20% FBS and 5 ng/ml GM-CSF. Fresh and frozenhuman bone marrow CD34⁺ cells were obtained from Stemcell Technologies(Vancouver, B.C., Canada). FACS analysis for CD34 antigen revealed apurity >98%.

Human Progenitor CD34⁺ Cell Cultures.

Human bone marrow CD34⁺ cells were grown in STEM-media (StemcellTechnologies), which includes Isocove modified Dulbecco's mediumsupplemented with human transferrin, insulin, bovine serine albumin,human low density lipoprotein and glutamine, in the presence of 100ng/ml human recombinant thrombopoietin (TPO) for the first 4 days,followed by a combination of 100 ng/ml TPO, IL3, and SCF (cytokinemixture CC-200, Stemcell Technologies). The initial cell density was100,000 cells/ml; three times a week, the cell density was adjusted to100,000 to 200,000 cells/ml. To increase the purity of the cells formicroarray analysis, cell sorting was performed at day 10 of culture.Cells were incubated on ice for 45 minutes with anti-human CD34⁺,anti-human CD41⁺, anti-human CD61⁺, and their respective isotypes. Afterwashing twice with PBS 3% FBS, cells were sorted using a FACS Ariasorting machine in bulk in two separate populations; CD34⁻ CD61⁺ andCD34⁺ CD61⁺ cells for culture and RNA extraction. The purity of thesorted populations was greater than 95%.

Megakaryocytes Characterization.

Cytospin preparations of CD34⁺ progenitors in culture were performed andstained with May-Grunwald Giemsa at different time points during themegakaryocytic differentiation induction. For FACS analysis, the primaryantibodies that were used were as follows: CD41A, CD61A, CD42B, and CD34with their respective isotypes (BD Pharmingen, San Diego, Calif.).Cytometric studies were performed as previously described (Tajima, S.,et al. (1996) J. Exp. Med. 184, 1357-1364) using a FACScalibur (BDBiosciences) and the CELLQUEST software (BD Biosciences).

RNA Extraction, Northern Blotting and miRNA Microarray Experiments.

Procedures were performed as described in detail elsewhere (Liu, C. G.,et al. (2002) Proc. Natl. Acad. Sci. USA 101, 9740-9744). Raw data werenormalized and analyzed in GENESPRING 7.2 software (zcomSiliconGenetics, Redwood City, Calif.). Expression data were median-centered byusing both GENESPRING normalization option and global mediannormalization of the BIOCONDUCTOR package (www.bioconductor.org) withsimilar results. Statistical comparisons were done by using theGENESPRING ANOVA tool, predictive analysis of microarray (PAM) and thesignificance analysis of microarray (SAM) software(www-stat.stanford.edu/˜tibs/SAM/index.html).

Reverse Transcriptase PCR(RT-PCR) and Real Time PCR.

Total RNA isolated with Trizol reagent (Invitrogen, Carlsbad, Calif.)was processed after DNAase treatment (Ambion, Austin, Tex.) directly tocDNA by reverse transcription using Superscript II (Invitrogen).Comparative real-time PCR was performed in triplicate. Primers andprobes were obtained from Applied Biosystems (Foster City, Calif.) forthe following genes: HOXA1, HOXA3, HOXB4, HOXB5, and HOXD10. Geneexpression levels were quantified by using the ABI Prism 7900 Sequencedetection system (Applied Biosystems). Normalization was performed byusing the 18S RNA primer kit. Relative expression was calculated byusing the computed tomography (CT) method. RT-PCR also was performed byusing the following oligonucleotide primers:

MAFB FW; 5′-AACTTTGTCTTGGGGGACAC-3′; (SEQ ID NO: 499) MAFB RW;5′-GAGGGGAGGATCTGTTTTCC-3′; (SEQ ID NO: 500) HOXA1 FW;5′-CCAGGAGCTCAGGAAGAAGA GAT-3′; (SEQ ID NO: 501) and HOXA1 RW;5′-CCCTCTGAGGCATCTGATTGGGTTT-3′. (SEQ ID NO: 502)

Real-Time Quantification of miRNAs by Stem-Loop RT-PCR.

Real time-PCR for pri-miRNAs 10a, miR15a, miR16-1, miR-130a, miR-20,miR-106, miR-17-5, miR-181b, miR-99a, and miR-126 were performed asdescribed (Chen, C., et al. (2005) Nucl. Acids Res. 33, e179. 18S wasused for normalization. All reagents and primers were obtained fromApplied Biosystems.

Bioinformatics.

miRNA target prediction of the differentially expressed miRNAs wasperformed by using TARGETSCAN (www.genes.mit.edu/targetscan), MIRANDA(www.mskc.miranda.org), and PICTAR (www.pictar.bio.nyu.edu) software.

Cell Transfection with miRNA Precursors.

miRNA precursors miR-10a and miR-130a were purchased from Ambion. Fivemillion K562 cells were nucleoporated by using Amaxa (Gaithesburg, Md.)with 5 μg of precursor oligonucleotides in a total volume of 10 ml. Theexpression of the oligonucleotides was assessed by Northern blots andRT-PCR as described.

Luciferase Reporter Experiments.

The 3′ UTR segments containing the target sites for miR-10a and miR-130afrom HOXA1 and MAFB genes, respectively, were amplified by PCR fromgenomic DNA and inserted into the pGL3 control vector (Promega, Madison,Wis.), by using the XbaI site immediately downstream from the stop codonof luciferase. The following oligonucleotide primer sets were used togenerate specific fragments:

MAFB FW 5′-GCATCTAGAGCACCCCAGAGGAGTGT-3′; (SEQ ID NO: 503) MAFB RW5′-GCATCTAGACAAGCACCATGCGGTTC-3′; (SEQ ID NO: 504) HOXA1 FW5′-TACTCTAGACCAGGAGCTCAGGAAGA-3′; (SEQ ID NO: 505) and HOXA1 RW5′-MCATTCTAGATGAGGCATCTGATTGGG-3′. (SEQ ID NO: 506)

We also generated two inserts with deletions of 5 bp and 9 bp,respectively, from the site of perfect complementarity by using theQuikChange XL-site directed Mutagenesis Kit (Stratagene, La Jolla,Calif.). Wild type (WT) and mutant insert were confirmed by sequencing.

Human chronic myeloid leukemia (CML) in megakaryoblastic crisis cellline (MEG-01) was cotransfected in six-well plates by usingLipofectamine 2000 (Invitrogen) according to the manufacturer's protocolwith 0.4 μg of the firefly luciferase report vector and 0.08 μg of thecontrol vector containing Renilla luciferase, pRL-TK (Promega). For eachwell, 10 nM of the premiR-130a and premiR-10a precursors (Ambion) wereused. Firefly and Renilla luciferase activities were measuredconsecutively by using the dual luciferase assays (Promega) 24 hoursafter transfection.

Western Blots.

Total and nuclear protein extracts from K562 cells transfected withmiR-10a and miR-130a, as well as CD34⁺ cells at different stages ofmegakaryocytic differentiation were extracted by using RIPA buffer orNuclear extraction Kit (Pierce, Rockford, Ill.). Protein expression wasanalyzed by Western blotting with the following primary antibodies: MAFB(Santa Cruz Biotechnology, Santa Cruz, Calif.), HOXA1 (R&D Systems,Minneapolis, Minn.), β-Actin and Nucleolin (Santa Cruz Biotechnology).Appropriate secondary antibodies were used (Santa Cruz Biotechnology).

EXAMPLE 1 miRNA Expression During in Vitro MegakaryocyticDifferentiation of CD34⁺ Progenitors

Using a combination of a specific megakaryocytic growth factor(thrombopoietin) and nonspecific cytokines (SCF and IL-3), we were ableto generate in vitro pure, abundant megakaryocyte progeny from CD34⁺bone marrow progenitors suitable for microarray studies (FIG. 4). TotalRNA was obtained for miRNA chip analysis from three different CD34progenitors at baseline and at days 10, 12, 14 and 16 of culture withcytokines. We initially compared the expression of miRNA between theCD34⁺ progenitors and the pooled CD34⁺ differentiated megakaryocytes atall points during the differentiation process. 17 miRNAs (Table 1) thatare sharply down regulated during megakaryocytic differentiation wereidentified. There were no statistically significant miRNAs upregulatedduring megakaryocytic differentiation. Using predictive analysis ofmicroarray (PAM), we identified 8 microRNAs that predictedmegakaryocytic differentiation with no misclassification error: miR-10a,miR-10b, miR-30c, miR-106, miR-126, miR-130a, miR-132, and miR-143. Allof these miRNAs, except miR-143, are included in the 17 miRNAsidentified by significance analysis of microarray (SAM). Northern blotsand real-time PCR for several miRNAs confirmed the results obtained bymiRNA chip analysis (FIG. 1).

Because we found mainly downregulation of miRNAs duringmegakaryocytopoiesis, we hypothesized that these miRNAs may unblocktarget genes involved in differentiation. In line with this hypothesis,miRNAs that are sharply downregulated in our system are predicted totarget genes with important roles in megakaryocytic differentiation.Among the transcription factors with well-known function inmegakaryocytopoiesis, RUNX-1 (Elagib, K. E., et al. (2003) Blood,101:4333-4341), Fli-1 (Athanasoiu, M., et al. (1996) Cell Growth Differ.7, 1525-1534), FLT1 (Casella, I., et al. (2003) Blood 101, 1316-1323),ETV6 (Hock, H., et al. (2004) Genes Dev. 18:2336-2341), TALI (Begley, C.G., and Green, A. R. (1999) Blood, 93:2760-2770), ETS1 (Jackers, P., etal. (2004) J. Biol. Chem. 279:52183-52190) and CRK (Lannutti, B. J., etal. (2003) Exp. Hematol. 12:1268-1274) are putative targets for severalmiRNAs downregulated in differentiated megakaryocytes. Moreover, each ofthese transcription factors has more than one miRNA predicted to be itsregulator. For example, RUNX1 (AML1) is predicted to be the target ofmiR-106, miR-181b, miR-101, let7d and the miR-17-92 cluster. Themultiplicity of miRNAs predicted to target AML1 suggests a combinatorialmodel of regulation.

We then looked at the temporal expression of miRNAs during themegakaryocytic differentiation process from CD34⁺ progenitors. Wefocused on miRNAs that have been described in hematopoietic tissues,such as miR-223, miR-181, miR-155, miR-142, miR-15a, miR-16, miR-106 andthe cluster of miR-17-92 (FIG. 5). We found sequential changes in theexpression of miR-223. Initially, miR-223 is downregulated duringmegakaryocytic differentiation, but after 14 days in culture, itsexpression returns to levels comparable with that of CD34 progenitors(FIG. 1C). The miR-15a and miR-16-1 cluster also follows the samepattern of expression as miR-223 (FIG. 1D), whereas miR-181b, miR-155,miR-106a, miR-17, and miR-20 were downregulated during differentiation(FIG. 6). The temporal variation of the expression of miR-223 andmiR-15a/mir-16-1 suggests a stage-specific function.

TABLE 2 miRNAs downregulated during in vitro CD34⁺ megakaryocyticdifferentiation. All differentially expressed miRNAs have q value <0.01(false-positive rate). Chromo- somal T-test Fold miRNA Location (†)Change Putative targets hsa-mir-010a* 17 q21 −9.10 50.00 HOXA1, HOXA3,HOXD10, CRK, FLT1 hsa-mir-126* 9q34 −2.73  8.33 CRK , EVI2, HOXA9, MAFB,CMAF hsa-mir-106* xq26.2 −2.63  2.86 TAL1, FLT1, SKI, RUNX1, FOG2, FLI,PDGFRA, CRK hsa-mir-010b* 2q31 −2.17 11.11 HOXA1, HOXA3, HOXD10, ETS-1,CRK, FLT1 hsa-mir-130a* 11q12 −2.08  4.76 MAFB, MYB, FOG2, CBFB, PDGFRA,SDFR1, CXCL12 hsa-mir-130a- 11q12 −2.07  7.69 NA± prec* hsa-mir-124a8q23 −1.81  2.78 TAL1, SKI, FLT1, FOG2, ETS-1, CBFB, RAF1, MYBhsa-mir-032- 9q31 −1.76  3.57 NA± prec hsa-mir-101 1p31.3 −1.75  3.33TAL1, CXCL12, MEIS1, MEIS2, ETS-1 RUNX1, MYB hsa-mir-30c 6q13 −1.71 2.56 CBFB, MAFG, HOXA1, SBF1, NCOR2, ERG hsa-mir-213* 1q31.3 −1.69 2.38 MAX- SATB2 hsa-mir-132- 17p13 −1.67  4.17 NA± prec hsa-mir-150*19q13.3 −1.63  5.26 MYB, SDFR1 hsa-mir-020 13q31 −1.62  2.17 TAL1, SKI,RUNX-1, FLT1, CRK, FOG2, RARB hsa-mir-339 7p22 −1.60  3.03 SKI, ETV6,GATA2, FLT1, RAP1B, JUNB, MEIS2 hsa-let-7a 9q22 −1.58  2.94 HOXA1,HOXA9, MEIS2, ITGB3, PLDN hsa-let-7d 9q22 −1.56  2.17 HOXA1, HOXD1,ITGB3, RUNX1, PDGFRA hsa-mir-181c 19p13 −1.55  2.50 RUNX-1, KIT, HOXA1,MEIS2, ETS-1 ETV6, PDGFRA hsa-mir-181b 1q31.3 −1.53  2.13 RUNX-1, KIT,ITGA3 , HOXA1, MEIS2, ETS-1, SDFR1, hsa-mir-017 13q31 −1.38  1.82 TAL1,SKI, FLT1, RUNX1, CRK, FOG1, ETS-1, MEIS1 †t test p < 0.05. *ThesemiRNAs were identified by PAM as predictors of a megakaryocytic classwith the lowest misclassification error. All, except miR-143 aredownregulated during megakaryocytic differentiation. NA±: miRNAprecursor sequence that does not contain the mature miRNA, therefore noputative target is shown.

EXAMPLE 2 MAFB Transcription Factor is a Target of miR-130a

By using three target prediction algorithms (TARGETSCAN(www.genes.mit.edu/targetscan), MIRANDA(www.microrna.org/miranda_new.html), and PICTAR(www.pictar.bio.nyu.edu)), we identified that miR-130a is predicted totarget MAFB, a transcription factor that is upregulated duringmegakaryocytic differentiation and induces the GPIIb gene, in synergywith GATA1, SP1 and ETS-1 (Sevinsky, J. R., et al. (2004) Mol. Cell.Biol. 24, 4534-4545). To investigate this putative interaction, first,we examined MAFB protein and mRNA levels in CD34⁺ progenitors atbaseline and after cytokine stimulation (FIG. 2A). We found that theMAFB protein is upregulated during in vitro megakaryocyticdifferentiation. Although the mRNA levels for MAFB by PCR increase withdifferentiation, this increase does not correlate well with theintensity of its protein expression. The inverse pattern of expressionof MAFB and miR-130a suggested in vivo interaction that was furtherinvestigated.

To demonstrate a direct interaction between the 3′ UTRs of MAFB withmiR-130a, we inserted the 3′ UTR regions predicted to interact with thismiRNA into a luciferase vector. This experiment revealed a repression ofabout ˜60% of luciferase activity compared with control vector (FIG.2B). As an additional control experiment, we used a mutated target mRNAsequence for MAFB lacking five of the complementary bases. As expected,the mutations completely abolished the interaction between miR-130a andits target 3′UTRs (FIG. 2B).

We also determined the in vivo consequences of overexpressing miR-130aon MAFB expression. The pre-miR-130a and a negative control weretransfected by electroporation into K562 cells, which naturally expressMAFB and lack miR-130a. Transfection of the pre-miR-130a, but not thecontrol, resulted in a decrease in the protein levels at 48 hours (FIG.2C). Northern blotting confirmed successful ectopic expression ofmiR-130a in K562 cells (FIG. 7).

EXAMPLE 3 MiR-10a Correlates with HOXB Gene Expression

It has been reported that in mouse embryos, miR-10a, miR-10b, andmiR-196 are expressed in HOX-like patterns (Mansfield, J. H., et al.(2004) Nature 36, 1079-1083) and closely follow their “host” HOX clusterduring evolution (Tanzer, A., et al. (2005) J. Exp. Zool. B Mol. Dev.Evol. 304B, 75-85). These data suggest common regulatory elements acrossparalog clusters. MiR-10a is located at chromosome 17q21 within thecluster of the HOXB genes (FIG. 8) and miR-10b is located at chromosome2q31 within the HOXD gene cluster. To determine whether the miR-10aexpression pattern correlates with the expression of HOXB genes, weperformed RT-PCR for HOXB4 and HOXB5, which are the genes located 5′ and3′, respectively, to miR-10a in the HOXB cluster. As shown in FIG. 8,HOXB4 and HOXB5 expression paralleled that of miR-10a, suggesting acommon regulatory mechanism.

EXAMPLE 4 MiR-10a Downregulates HOXA1

We determined by miRNA array and Northern blot that miR-10a is sharplydown-regulated during megakaryocytic differentiation. Interestingly, wefound several HOX genes as putative targets for miR-10a (Table 2). Wethus investigated whether miR-10a could target a HOX gene. We performedreal-time PCR for the predicted HOX targets of miR-10: HOXA1, HOXA3, andHOXD10. After normalization with 18S RNA, we found that HOXA1 mRNA isupregulated 7-fold during megakaryocytic differentiation compared withCD34 progenitors (FIG. 3A; see also FIG. 9). HOXA1 protein levels werealso upregulated during megakaryocytic differentiation (FIG. 3B). Theseresults are in sharp contrast with the downregulation of miR-10a inmegakaryocytic differentiation, suggesting that miR-10a could be aninhibitor of HOXA1 expression. To demonstrate a direct interaction ofmiR-10a and the 3′ UTR sequences of the HOXA1 gene, we carried out aluciferase reporter assay as described in Material and Methods. When themiRNA precursor miR-10a was introduced in the MEG01 cells along with thereporter plasmid containing the 3′ UTR sequence of HOXA1, a 50%reduction in luciferase activity was observed (FIG. 3C). The degree ofcomplementarity between miR-10a and the HOXA1 3′ UTR is shown in FIG.3D, as predicted by PICTAR (www.pictar.bio.nyu.edu).

To confirm in vivo these findings, we transfected K562 cells with thepre-miR-10a precursor using nucleoporation and measured HOXA1 mRNAexpression by RT-PCR and HOXA1 protein levels by Western blotting.Successful ectopic expression of miR-10a was documented by Northern Blot(FIG. 3E). A significant reduction at the mRNA and protein levels forHOXA1 was found for K562 cells transfected with the miR-10a precursorbut not with the negative control (FIGS. 3F and 3G). These data indicatethat miR-10a targets HOXA1 in vitro and in vivo.

It has been reported that miR-196 induces cleavage of HOXB8 mRNA,pointing to a posttranscriptional restriction mechanism of HOX geneexpression (Yekta, S., et al. (2004) Science, 304:594-596). Contrary tothe miR-196-HOXB8 interaction, where an almost perfect complementarityexists, the degree of pairing between miR-10a and the human HOXA1 3′ UTRis suboptimal (FIG. 4). Although our results indicated target mRNAdegradation, further studies are needed to determine whether cleavage ortranslational repression is the primary mechanism of downregulation ofthe HOXA1 gene in this system. A previous study using microarrayanalysis showed that a large number of target mRNA genes aredownregulated by miRNA at the level of transcription (Lim, L. P., et al.(2005) Nature: 433, 769-771). These data raise the question whethertarget degradation is a consequence of translational repression andsubsequent relocalization of the miR-target complexes to cytoplasmicprocessing bodies or is a primary event (Pillai, R. (2005) RNA 11,1753-1761).

EXAMPLE 5 miRNA Profiling in Acute Megakaryoblastic Leukemia (AMKL) CellLines

After the identification of the microRNA expression profile of CD34⁺cells during megakaryocytic differentiation, we then investigated miRNAexpression in AMKL cell lines with the goal to identify differentiallyexpressed miRNAs that could have a pathogenic role in megakaryoblasticleukemia. We initially compared miRNA expression in four AMKL cell lineswith that of in vitro CD34⁺-differentiated megakaryocytes. Usingsignificance analysis of microarray (SAM), we identified 10 miRNAsupregulated in AMKL cell lines compared with that of CD34 invitro-differentiated megakaryocytes (Table 3; see also Table 4). ThesemiRNAs are as follows (in order of the fold increase with respect todifferentiated megakaryocytes): miR-101, miR-126, miR-99a, miR-99-prec,miR-106, miR-339, miR-99b, miR-149, miR-33 and miR-135. Results werevalidated by RT-PCR as shown in FIG. 10. Using PAM, we compared miRNAexpression in CD34⁺ cells with in vitro-differentiated megakaryocytesand AMKL cell lines (FIG. 10). Interestingly, we found five miRNAsinvolved in the megakaryocytic differentiation signature (miR-101,miR-126, miR-106, miR-20, and miR-135) that were upregulated in theleukemic cell lines (Tables 3, 5 and 6). Whether this profile representsmerely a differentiation state of the cells or has a truly pathogenicrole remains to be elucidated. Supporting the second hypothesis,miR-106, miR-135, and miR-20 are predicted to target RUNX1, which is oneof the genes most commonly associated with leukemia (Nakao, M., et al.(2004) Oncogene 125, 709-719). Moreover, mutations of RUNX1 have beendescribed in familial thrombocytopenias with a propensity to developacute myeloid leukemia (Song, W. J., et al. (1999) Nat. Genet. 23,166-175).

TABLE 3 microRNAs upregulated in acute megakaryoblastic cell linescompared with in vitro-differentiated megakaryocytes. Chro- mosomal ttest Fold microRNA Location Score Change Putative Targets hsa-mir-1011p31.3 6.14 11.85 MEIS2, RUNX1, ETS-1, C-MYB, FOS, RARB, NFE2L2hsa-mir-126 9q34 4.91 11.97 V-CRK hsa-mir-099a 21q21 3.30 6.83 HOXA1,EIF2C, FOXA1 hsa-mir-099b- 21q21 2.85 7.59 NA prec hsa-mir-106 xq26.22.79 3.33 FLT1, SKI, E2F1, NCOA3, PDGFRA, CRK hsa-mir-339 7p22 2.58 3.36HOXA1, FLT1, PTP4A1, RAP1B hsa-mir-099b 19q13 2.46 4.19 HOXA1, MYCBP2hsa-mir-149 2q37 2.29 3.53 RAP1A, MAFF, PDGFRA, SP1, NFIB hsa-mir-0332q13 2.27 3.23 PDGFRA, HIF1A, MEIS2 hsa-mir-135 3p21 2.12 3.97 SP1,HIF1A, SP3, HNRPA1, HOXA10, RUNX1

All the miRNAs have a q value <0.01 (false discovery rate).

The same miRNAs, except miR-339 and miR-149, were found by using PAM topredict a megakaryoblastic leukemia class with no misclassificationerror.\

The results described herein demonstrate that there is a downregulationof miRNAs during megakaryocytopoiesis. Hypothetically, thedownregulation of miRNAs unblocks target genes involved indifferentiation. In line with this hypothesis, miRNAs that are sharplydownregulated in our system are predicted to target genes with importantroles in megakaryocytic differentiation. Thus, we have shown thatmiR-130a targets MAFB and miR-10a modulates HOXA1. The fact that wefound several differentially expressed miRNAs during differentiation andleukemia that are predicted to target HOXA1 suggests a function forHOXA1 in megakaryocytopoiesis. Loss and gain studies will ultimately beneeded to define the role of HOXA1 in this differentiation process. Ourfindings delineate the expression of miRNAs in megakaryocyticdifferentiation and suggest a role for miRNA modulation of this lineageby targeting megakaryocytic transcription factors. Furthermore, inmegakaryoblastic leukemia cell lines, we have found inverse expressionof miRNAs involved in normal megakaryocytic differentiation. These dataprovide a starting point for future studies of miRNAs inmegakaryocytopoiesis and leukemia.

TABLE 4 Signature of megakaryocytic differentiation. CD34 MegakaryocyticmicroRNA Expression Expression hsa-mir-010a up Down hsa-mir-126 up Downhsa-mir-130a-prec up Down hsa-mir-010b up Down hsa-mir-106 up Downhsa-mir-130a up Down hsa-mir-132 up Down hsa-mir-30c up Downhsa-mir-143-prec Down up

PAM selected microRNAs with a very low misclassification error.

TABLE 5 Signature of megakaryoblastic leukemia cell lines Level ofExpression t test Fold in AML MicroRNA Score Change M7 Putative Targetshsa-mir-101- 6.14 11.85 up MEIS2, RUNX1, C-MYB, FOS, RARb, NFE2L2hsa-mir-126 4.91 11.97 up V-CRK hsa-mir-099a 3.30 6.83 up HOXA1, EIF2C,FOXA1 hsa-mir-095 up SHOX2 hsa-mir-033 2.27 3.23 up PDGFRA, HIF1A, MEIS2hsa-mir-135 2.12 3.97 up SP1, HIF1A, SP3, HNRPA1, HOXA10, RUNX1hsa-mir-099b 2.85 7.59 up HOXA1, MYCBP2 hsa-mir-339 2.58 3.36 up HOXA1,FLT1, PTP4A1, RAP1B hsa-mir-106 2.79 3.33 up HOXA1, EIF2C, FOXA1hsa-mir-124a 2.07 2.78 up SDFR1, RXRa hsa-mir-155 down ETS-1 hsa-mir-0202.00 3.09 up TAL1 SKI, RUNX-1, FLT1, CRK, FOG2, RARB hsa-mir-025 1.984.24 up GATA2, hsa-mir-140 down GATA1

PAM selected microRNAs. The fold change of miRNA expression is shownalongside t test score (SAM) and putative targets.

Table 6 Three class analysis showing the different regulated microRNAsamong the three cell types: CD34⁺ progenitors, acute megakaryoblasticleukemia cell lines (AMKL) and in vitro-differentiated megakaryocytes.

TABLE 6 AML In Vitro- Chromo- M7 cell differentiated somal CD34⁺ linesMegakaryocytes microRNA Location Score score Score hsa-mir-010a 17q211.0198   0    −0.3562 hsa-mir-101 1p31.3 0      0.814  −0.432 hsa-mir-126 9q34 0.0621   0.4882 −0.4514 hsa-mir-099a 21q21 0     0.4685 −0.2875 hsa-mir-033 22q13 0      0.4258 −0.2294 hsa-mir-0954p16 0      0.4142 −0.3567 hsa-mir-010b 2q31 0.3308   0      0   hsa-mir-155 21q21 0    −0.3217   0    hsa-mir-130a 11q12 0.2755   0     0    hsa-let-7d 9q22 0.263  −0.274    0    hsa-mir-099b-prec 21q210      0.266  −0.1078 hsa-mir-135-2-prec 12q23 0      0.2279 −0.2566hsa-mir-339 7p22 0      0.2456 −0.1176 hsa-mir-099b 19q13 0      0.2275−0.1025 hsa-mir-106 xq26 0      0.0575 −0.1891 hsa-let-7c 21q21 0.0289−0.1753   0    hsa-mir-148 7p15 0    −0.1748   0    hsa-mir-132-prec17p13 0.1721   0      0    hsa-mir-020 13q31 0      0.0374 −0.1509

There are three patterns of miRNA expression among the three differentcell types. The first pattern is defined by miRNA highly expressed inCD34⁺ cells and downregulated in AMKL and differentiated megakaryocytes.miR-10a and miR-130a follow this pattern of expression; however, miR-10ais upregulated in AMKL relative to differentiated megakaryocytes. Thesecond pattern is miRNA that is upregulated in AMKL, downregulated inCD34⁺ cells and differentiated megakaryocytes and includes the followingmiRNAs: miR-126, miR-99, miR-101, let 7A, and miR-100. The last twomiRNAs are equally expressed in CD34⁺ and differentiated megakaryocytes,rather than showing a gradual decline in expression, as evidenced bymiR-126, miR-99 and miR-101. The last pattern includes miRNA-106 andmiRNA-135-2, which are upregulated in CD34⁺ cells and AMKL, but low indifferentiated megakaryocytes.

MicroRNAs are a highly conserved class of non-coding RNAs with importantregulatory functions in proliferation, apoptosis, development anddifferentiation. As described herein, to discover novel regulatorypathways during megakaryocytic differentiation, we performed microRNAexpression profiling of in vitro-differentiated megakaryocytes derivedfrom CD34⁺ hematopoietic progenitors. One major finding wasdownregulation of miR-10a, miR-126, miR-106, miR-10b, miR-17 and miR-20.Without wishing to be bound to any theory, it is believed that thedownregulation of microRNAs unblocks target genes involved indifferentiation. It was confirmed in vitro and in vivo that miR-130atargets the transcription factor MAFB, which is involved in theactivation of the GPIIB promoter, a key protein for platelet physiology.In addition, it was shown that miR-10a expression in differentiatedmegakaryocytes is inverse to that of HOXA1, and HOXA1 is a direct targetof miR-10a. Finally, the microRNA expression of megakaryoblasticleukemic cell lines was compared to that of in vitro-differentiatedmegakaryocytes and CD34⁺ progenitors. This analysis revealedupregulation of miR-101, miR-126, miR-99a, miR-135, and miR-20 in thecancerous cell line. The data and results described herein delineate theexpression of microRNAs during megakaryocytopoiesis and demonstrate aregulatory role of microRNAs in this process by targeting megakaryocytictranscription factors.

The relevant teachings of all publications cited herein that have notexplicitly been incorporated by reference, are incorporated herein byreference in their entirety. While this invention has been particularlyshown and described with references to preferred embodiments thereof, itwill be understood by those skilled in the art that various changes inform and details may be made therein without departing from the scope ofthe invention encompassed by the appended claims.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to the particular embodimentdisclosed herein contemplated for carrying out this invention, but thatthe invention will include all embodiments falling within the scope ofthe claims.

1. A method of diagnosing acute megakaryoblastic leukemia in a subject,comprising: i) comparing the level of at least one miR-155 gene productin a subject sample to control; and ii) diagnosing acutemegakaryoblastic leukemia in the event that the at least one miR-155gene product level is lower in the sample compared to control.
 2. Themethod of claim 1, wherein the control is the level of the at least onemiR-155 gene product from a subject that does not have a acutemegakaryoblastic leukemia disorder.
 3. The method of claim 1, whereinthe subject is a human.
 4. The method of claim 1, wherein the control isthe level of the at least one miR-155 gene product from a referencesample comprising non-differentiating megakaryocyte progeny and/ormegakaryocytes.
 5. The method of claim 1, which further comprises: i)comparing the level of at least one additional miR gene product in asubject sample to control, wherein the at least one additional miR geneproduct is selected from the group consisting of: miR-101; miR-126;miR-99a; miR-99-prec; miR-106; miR-339; miR-99b; miR-149; miR-33;miR-135; and miR-20; and ii) diagnosing acute megakaryoblastic leukemiain the event that the at least one additional miR gene product level ishigher in the sample compared to control.
 6. A method of diagnosingacute megakaryoblastic leukemia in a subject, comprising: i) comparingthe level of at least one miR-140 gene product in a subject sample tocontrol; and ii) diagnosing acute megakaryoblastic leukemia in the eventthat the at least one miR-140 gene product level is lower in the samplecompared to control.
 7. The method of claim 6, wherein the control isthe level of the at least one miR-140 gene product from a subject thatdoes not have a acute megakaryoblastic leukemia disorder.
 8. The methodof claim 6, wherein the subject is a human.
 9. The method of claim 6,wherein the control is the level of the at least one miR-140 geneproduct from a reference sample comprising non-differentiatingmegakaryocyte progeny and/or megakaryocytes.
 10. The method of claim 6,which further comprises: i) comparing the level of at least oneadditional miR gene product in a subject sample to control, wherein theat least one additional miR gene product is selected from the groupconsisting of: miR-101; miR-126; miR-99a; miR-99-prec; miR-106; miR-339;miR-99b; miR-149; miR-33; miR-135; and miR-20; and ii) diagnosing acutemegakaryoblastic leukemia in the event that the at least one miR geneproduct level is higher in the sample compared to control.
 11. A methodof diagnosing-acute megakaryoblastic leukemia in a subject, comprising:i) comparing the levels of at least one miR-155 gene product and atleast one miR-140 gene product in a subject sample to control; and ii)diagnosing acute megakaryoblastic leukemia in the event at least onemiR-155 gene product and at least one miR-140 gene product levels arelower in the sample compared to control.
 12. The method of claim 11,wherein the control is the level of the at least one miR-155 geneproduct from a subject that does not have a acute megakaryoblasticleukemia disorder.
 13. The method of claim 11, wherein the subject is ahuman.
 14. The method of claim 11, wherein the control is the level ofthe at least one miR-155 gene product from a reference sample comprisingnon-differentiating megakaryocyte progeny and/or megakaryocytes.
 15. Themethod of claim 11, which further comprises: i) comparing the level ofat least one additional miR gene product in a subject sample to control,wherein the at least one additional miR gene product is selected fromthe group consisting of: miR-101; miR-126; miR-99a; miR-99-prec;miR-106; miR-339; miR-99b; miR-149; miR-33; miR-135; and miR-20; and ii)diagnosing acute megakaryoblastic leukemia in the event that the atleast one miR gene product level is higher in the sample compared tocontrol.